Importance of Loop L1 Dynamics for Substrate Capture and Catalysis

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Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa D-Arginine Dehydrogenase Daniel Ouedraogo, Michael Souffrant, Sheena Vasquez, Donald Hamelberg, and Giovanni Gadda Biochemistry, Just Accepted Manuscript • Publication Date (Web): 26 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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Biochemistry

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa D-Arginine Dehydrogenase

Daniel Ouedraogo1, Michael Souffrant1, Sheena Vasquez1,5, Donald Hamelberg1, 3, 4, and Giovanni Gadda1, 2, 3, 4, *

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Department of Chemistry, 2Department of Biology, 3Center for Diagnostics and Therapeutics,

Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302, United States 5

Current address: University of Georgia, Athens, Georgia 30602

Running Title: Importance of Loop Dynamics in Substrate Capture and Catalysis

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ABSTRACT Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control substrate access to the active site, and to position residues for substrate binding and catalysis. In D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations of loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have higher probabilities than wild-type PaDADH of loop L1 to be in open conformations, yielding increased solvent exposure of the active site. In agreement, flavin fluorescence intensity was ~2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the kcat/Km values with D-arginine were ~13-fold lower than in wild-type PaDADH. Moreover, the pH-profiles for the kcat value with D-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate D-leucine in the reductive half-reaction of the variant enzymes. Taken together the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

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Biochemistry

The motion of amino acid residues in mobile loops has been recognized to be relevant to catalysis in several enzymes.1-4 Mobile loops located at the entrance of the active site pocket typically act as gates to control access and exit of substrates and products as well as to shield the active site from the bulk solvent for catalysis.5-8 Although most of the residues in loops do not act as catalytic per se, they often participate in conformational changes required to position residues that are relevant to substrate binding and catalysis.9-12 In lactate oxidase, Y215 functions as a latch that fastens a lid over the active site for the optimal reduction and oxidation of the enzyme-bound flavin.13 In phosphoenolpyruvate carboxykinase, E89 in the R-loop region of the enzyme helps to coordinate the interactions between the substrate and the substrate binding-site that results in the optimization of the phosphoryl transfer distance required in catalysis.14 In dihydrofolate reductase, the M20 loop fluctuates between interacting with the dihydrofolate substrate and occupying a portion of the NADP(H) binding pocket in the active site on time scales that are relevant to catalysis.15 In triosephosphate isomerase, the closure of loop 6 results in the desolvation of the active site base E165 and the sequestration of the substrate from the bulk solvent that are required for catalysis.4 In D-arginine dehydrogenase, the crystallographic data reveal that two peptidyl regions including residues 45-47 and 50-56 in the active site loop L1 could adopt two distinct conformations depending on whether the substrate is present in the active site of the enzyme or not (Figure 1).16 However, no studies have been carried out to establish a link between the dynamics and conformational changes undertaken by the two peptidyl regions of loop L1 as well as their implication in the catalytic function of the enzyme.

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Figure 1. Open and closed conformations of loop L1 in wild-type PaDADH (PDB: 3NYC). Panels A and B show the overall structure of the enzyme with the flavin-binding and substratebinding domains and loop L1 in the open (orange) and closed (green) conformations. IAR is the iminoarginine product bound in the active site. Panel C shows the distances between the flavin N5 atom and the C4 atom of Y53, and between the αC atom of Y53 in the open and closed conformations. Note that in the closed conformation there is a hydrogen bond interaction between the hydroxyl O atom of S45 and the 2’-OH of the ribityl moiety of the FAD cofactor.

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Biochemistry

Scheme 1: D-Arginine Oxidation by PaDADH

D-Arginine dehydrogenase (PaDADH; E.C.1.4.99.6) from Pseudomonas aeruginosa is an FAD-dependent enzyme that catalyzes the oxidation of D-arginine to α-iminoarginine, which non-enzymatically hydrolyzes to α-ketoarginine and ammonia (Scheme 1).17-19 The physiological substrate that oxidizes the flavin in turnover is not known, but the enzyme does not utilize molecular oxygen thereby it acts as a true dehydrogenase.19 PaDADH participates with Larginine dehydrogenase in the racemization of arginine, allowing for the growth of P. aeruginosa on D-arginine as the sole carbon and nitrogen source.19 Structurally, PaDADH belongs to a family of flavin-dependent enzymes that oxidize amino acids, which includes among others D-amino acid oxidase, dimethyl glycine oxidase, glycine oxidase and sarcosine oxidase.20-26 A characteristic of PaDADH is its wide substrate specificity, being able to utilize as a substrate all the Damino acids with the exception of D-aspartate and D-glutamate.16 The cationic D-arginine and D-lysine are the preferred substrates for the enzyme, with kcat/Km values of 105-106 M-1s-1,16 primarily due to an electrostatic interaction of their side chain with E87 in the active site of the enzyme.27 The role of other residues in the active site of the enzyme has been elucidated with sitedirected mutagenesis. H48, through the action of two intervening water molecules, is important 5


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for the optimal orientation of the D-amino acid substrate in the enzyme-substrate complex that proceeds to catalysis.27 Y53 and Y249 stabilize the transition state for the transfer of the hydride ion from the substrate αC atom to the flavin N5 atom.28 Y53, in the 50-56 peptidyl region of loop L1 that is adjacent to the active site entrance, was proposed to act as a gate granting access of the substrate to the active site of the enzyme based on the observation that its side chain shares a hydrogen bond with the carboxylate group of the α-iminoarginine product in the enzymeproduct complex, and it points outward in the free enzyme structure.16 The distance between the C4 atom of Y53 and the N5 atom of the flavin was ~6.9 Å for the product-bound conformation and ~14.3 Å for the ligand-free conformation (Figure 1). Concomitantly, in the 45-47 peptidyl region of loop L1, which is located at the FAD-binding domain, only the S45 and A46 residues adopt major conformational changes. In the ligand-free conformation the side chains of S45 and A46 point away from the FAD (Figure 1). In the product-bound conformation, instead, the side chain of A46 moves closer to the flavin while the side chain of S45 interacts with the 2’-OH group of the ribityl moiety of the flavin (Figure 1).16 Thus, the S45/A46 dipeptide was proposed to act as a switch with possible implications for the catalytic function of the enzyme.16 In this study, the S45 and A46 residues in PaDADH were individually mutated to the smaller residues alanine and glycine, respectively, to alter the dynamics of loop L1. The resulting enzyme variants were characterized using molecular dynamics, steady-state and rapid reaction kinetics, pH effects, and fluorescence spectroscopy to investigate the importance of the S45/A46 switch in loop L1 for substrate capture and catalysis in PaDADH.

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Biochemistry

EXPERIMENTAL PROCEDURES Materials. Escherichia coli Rosetta(DE3)pLysS was purchased from Novagen (Madison, WI). The QIAprep Spin Miniprep Kit and the QIAquick Polymerase Chain Reaction (PCR) Purification Kit were obtained from Qiagen (Valencia, CA). Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA) and DpnI was obtained from New England BioLabs (Ipswich, MA). Oligonucleotides for site-directed mutagenesis were purchased from Sigma Genosys (The Woodlands, TX). Phenazine methosulfate (PMS) was from Sigma-Aldrich (St. Louis, MO). DAmino acids were obtained from Alfa-Aesar (Ward Hill, MA). All other reagents used were obtained at the highest purity commercially available. Molecular Dynamics. In all simulations, the initial coordinates were taken from the PDB entry 3NYC with a resolution of 1.06 Å for wild-type PaDADH. This entry presents the enzyme in both the open and closed conformations. All the residues present in the open conformation and the imino product of the enzymatic reaction were removed from the PDB file to ensure that the closed conformation was the starting point for the simulations. The S45A and A46G enzyme variants were produced in silico from the structure of the wild-type enzyme in the closed conformation. Amber 14 software29 was used to run all simulations with the ff14sb modified force field parameters of Cornell et al.30 by Maier et al.31 The AmberTools xleap program was used to construct the appropriate system required for each MD simulation. The parameters of the FAD cofactor originated from the general AMBER force field (GAFF).32 Each system was properly solvated with a 10 Å TIP3P octahedron box, followed by the addition of sodium ions for neutralization purposes. All simulations were minimized for 1000 steps with harmonic constraints of a 100 kcal x mol-1 x Å-2 on PaDADH and the FAD cofactor. Afterwards, all systems underwent three rounds of equilibration with harmonic constraints in decreasing order of 50 kcal x mol-1 x

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Å-2, 25 kcal x mol-1 x Å-2, and 0 kcal x mol-1 x Å-2 on the enzyme and its cofactor. All equilibrations were processed at a constant temperature of 300 K and pressure of 1 bar. The temperature regulation was maintained using the Langevin thermostat through a 1 ps-1 collision frequency. A time step of 2 fs was used to solve Langevin equation of motion, however, the first equilibrated molecular dynamics step was carried using 1 fs time step. Long-range non-bonded electrostatic interactions were evaluated using the particle mesh Ewald (PME) method,33 and all non-bonded interactions were subjectied to a cutoff of 9 Å. The SHAKE algorithm was used to constrained all bonds involving hydrogen atoms.34 The molecular dynamics simulation of each variant was carried out for 1.1 µs, in which case, snapshots of the first 0.1 µs were discarded as equilibration phase. The remaining 1.0 µs was the only segment of the trajectory considered for analysis. The RMSD of PaDADH was evaluated in respect to the backbone atoms of each residue within the enzyme, whereas the RMSD of the FAD cofactor targeted the flavin component, in which case, the flavin N1, N5, and C9 atoms were taken into consideration. This approach was plausible due to the planarity of the flavin component in regards to the the N1, N5, and C9 atoms. A localized FAD cofactor fluctuation enabled the distance analyses between the N5 atom of FAD and the C4 atom of Y53 (Figure 2). RMSD assessments, distance analyses, and probability distributions were evaluated through PTRAJ,35 in which case, graphical representations of the results were plotted using xmgrace. Site-Directed Mutagenesis, Protein Expression and Purification. The S45A and A46G enzyme variants of PaDADH were generated by mutagenic polymerase chain reaction (PCR). The plasmid pET20b(+)/PA3863 harboring the wild-type gene (dauA) encoding for Darginine dehydrogenase was used as a template. The forward primer for the S45A mutation was 5’-CACTCCACCGGCCGCGCCGCCGCGCACTACAC-3’ and for the A46G mutation was 5’-

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Biochemistry

TCCACCGGCCGCTCCGGCGCGCACTACACGGTGGC-3’. The underlined portions on the oligonucleotides represent the mutated sites. Dimethyl sulfoxide was added to the PCR reaction mixture at a final concentration of 5% to ensure proper separation of the highly GC-rich, doublestranded DNA template. Site-directed mutagenesis amplicons were purified as per the manufacturer’s instructions using the QIAquick PCR Purification Kit before treating the samples with DpnI at 37 oC for 2 h and transformation into E. coli strain DH5α. Each mutation was confirmed through sequencing the gene using an Applied Biosystems Big Dye Kit on an Applied Biosystems model ABI377 DNA Sequencer, at the DNA Core Facility of Georgia State University. The S45A and A46G enzyme variants of PaDADH were expressed in E. coli strain Rosetta(DE3)pLysS and purified to homogeneity as previously described for the PaDADH wild-type enzyme in the presence of 10% (v/v) glycerol in order to minimize enzyme instability.36 The enzymes were stored at -20 oC in 20 mM TRIS-Cl, pH 8.0, and 10% glycerol, and were found to be active for at least six months. UV-Visible Absorption and Fluorescence Spectroscopy. The UV-visible absorption spectra of the S45A and A46G enzyme variants of PaDADH were acquired with an Agilent Technologies model HP 8453 PC diode-array spectrophotometer equipped with a thermostated water bath. The enzymes were prepared fresh by gel filtration through PD-10 desalting columns (General Electric, Fairfield, CT) just prior to being used. The extinction coefficients of the enzyme-bound FAD in the S45A and A46G enzyme variants were determined in 20 mM sodium phosphate, pH 7.0, after incubation of the enzymes with 4 M urea at 40 °C for 1 h, based upon an ε450 value of 11.3 mM-1 cm-1 for free FAD and the method published by Whitby et al.37 The fluorescence emission spectra of the S45A and A46G enzyme variants were recorded in 20 mM sodium phosphate, pH 7.0 and 15 °C, with a Shimadzu model RF-5301 PC spectrofluorometer us-

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ing a 1 cm path length quartz cuvette. All fluorescence spectra were corrected by subtracting the corresponding blanks to account for Rayleigh and Raman scattering. The samples at a concentration of 3.5 µM enzyme-bound flavin were excited at the low energy peak of the UV-visible absorption spectrum and emission scans were determined from 475 nm to 600 nm. The fluorescence emission of free FAD was determined on a flavin sample extracted from the FADdependent HupX enzyme from Streptococcus pyogenes (Elvira Romero and Giovanni Gadda, unpublished results) rather than using commercially available FAD to avoid commonly found contamination due to FMN. Steady-State Kinetics pH-Profiles. The steady-state kinetic parameters with D-arginine as a substrate for the S45A and A46G enzyme variants of PaDADH were determined using PMS as electron acceptor and the method of the initial rates.36, 38 PaDADH is a true dehydrogenase and as such it does not react with molecular oxygen;36 thus, the spontaneous oxidation of the PMS reduced enzymatically by PaDADH by molecular oxygen was followed with a Clark-type oxygen electrode. The final concentrations of enzyme in 1 mL reaction mixtures ranged from 5 nM to 10 µM and those of D-arginine ranged from 0.01 mM to 300 mM depending upon the pH used. In all cases, attempts were made to ensure that the Km value was within the range of the substrate concentrations used. For those pH values for which this was not possible, only the kcat/Km values were reported. The enzymatic assays were carried out at 25 oC in 20 mM sodium phosphate or 20 mM piperazine depending on the pH value. Substrate solutions were prepared in buffer and the pH values were readjusted after dissolving the amino acid substrates. The concentration of PMS was kept fixed at 1 mM since the Km value for PMS in the wild-type enzyme was previously determined to be ~10 µM, irrespective of the substrate used.36 To ensure that both the S45A and the A46G enzyme variants were fully saturated with PMS throughout the pH

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Biochemistry

range investigated, the steady-state kinetic parameters at the pH extremes were determined also at 1.5 mM PMS. Reductive Half-Reaction. The reductive half-reactions were carried out using an SF61DX2 Hi-Tech KinetAsyst performance stopped-flow spectrophotometer in 20 mM sodium pyrophosphate, pH 10.0 and 25 oC. With both the S45A and A46G enzyme variants flavin reduction occurred almost completely within the mixing time of the stopped-flow spectrophotometer (i.e., 2.2 ms) when D-arginine was the substrate, as previously established for wild-type PaDADH and the Y53 and Y249 enzyme variants.28, 39 The reductive half-reaction of the S45A and A46G variant enzymes was thus investigated with D-leucine as a reducing substrate, as previously done with wild-type PaDADH and the Y53 and Y249 enzyme variants. Due to a small but not negligible reaction of the reduced enzyme-bound flavin with molecular oxygen in both the S45A and A46G enzyme variants (data not shown), the reductive half-reactions had to be carried out anaerobically. Anaerobiosis of the instrument was obtained by overnight incubation with a solution containing 5 mM glucose and 1 µM glucose oxidase in sodium pyrophosphate, pH 6.0. The enzyme was passed through a desalting PD-10 column equilibrated with 20 mM sodium pyrophosphate, pH 10.0, transferred in a tonometer and subjected to 20 cycles of degassing by applying vacuum and flushing with argon. The syringes containing the buffer or the substrate Dleucine in buffer were flushed for 30 min with argon before mounting onto the stopped-flow spectrophotometer. To ensure complete removal of traces of oxygen, glucose (2 mM) and glucose oxidase (0.5 µM) were present in the buffer, enzyme and substrate solutions. The maintain pseudo first-order conditions, the final concentration of D-leucine after mixing ranged from 1 mM to 60 mM and the concentration of the enzyme was ~10 µM.

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Data Analysis. Kinetic data were fit with the KaleidaGraph software (Synergy Software, Reading, PA) and the Kinetic Studio Software Suite Enzfitter (Hi-Tech Scientific, Bradford on Avon, U.K). The steady-state kinetic parameters at varying concentration of the amino acid substrate and fixed concentration of PMS were determined by using the Michaelis-Menten equation for a single substrate. The time-resolved flavin reductions were fit to eq 1, which describes a single exponential process for flavin reduction. Here kobs represents the observed first-order rate constant for the reduction of the enzyme-bound flavin at any given concentration of the substrate associated with the absorption changes at 450 nm, A represents the absorbance at 450 nm at any given time, B is the amplitude of the absorption changes, t is time and C is the absorbance at infinite time that accounts for the non-zero absorbance of the fully reduced enzyme-bound flavin. = exp− +

(1)

The kinetic parameters of the reductive half-reaction were determined by using eq 2, which defines a hyperbolic saturation of the enzyme with D-leucine with a y-intercept value of zero. Here kobs represents the observed first-order rate constant for the reduction of the enzymebound flavin at any given substrate concentration (S), kred is the limiting first-order rate constant for flavin reduction at saturating substrate concentrations, and Kd is the apparent equilibrium constant for the dissociation of the enzyme-substrate complex into free substrate and enzyme. The use of an equation that defines a hyperbolic saturation with a finite y-intercept yielded a value not significantly different from zero for the y-intercept.

=

(2)

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Biochemistry

The pH-profiles of the kcat/Km value for D-arginine were fit to eq 3, which describes a profile that increases with increasing pH with a slope of +2, a limiting value (kcat/Km)H at high pH and two indistinguishable pKa values. log ⁄ = log

"#$ ⁄% &

(3)

,

''()*# +)&

The pH-profiles of the kcat value for D-arginine were best fit to eq 4 instead of eq 5. Eq 4 describes a profile that increases with increasing pH to a limiting value (kcat)H to yield three pH dependent terms containing a hollow that is defined by β = 1 + k3/k2 and Kα = (k1/k13)(1+k14/k15)K1 (Scheme 2). Here, pKe is the intrinsic pKa value for kcat, β reflects the stickiness of the substrate, and Kα describes the relative rates of the kinetic steps associated with the ESH+ complex equilibrium;40 Eq 5 describes a profile that increases with increasing pH with slope of +1 and a limiting value, (kcat)H, at high pH. log = log log = log

"#$ & ∗ .''()*/ +)& 0 ''()* +)& .''(1)*/ +)& 0 "#$ &

(4) (5)

''()*# +)&

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RESULTS Molecular Dynamics. Molecular dynamic simulations were carried out on the S45A and A46G enzyme variants as well as on wild-type PaDADH to reveal whether the conformational change of the S45/A46 dipeptide is correlated with the movement of Y53 in loop L1. The RMSD assessments of the wild-type PaDADH and the two enzyme variants indicated that the enzymes and their cofactors were relatively localized over 1 µs in comparison to their frame of reference at 0.1 µs (Figure 2). This is consistent with absence of independent movement of the FAD with respect to the protein. Instead, the time courses of the distance between the C4 atom of Y53 and the flavin N5 atom were consistent with the three enzymes rapidly sampling a few conformational states over the time frame of the simulation, but with significantly different probabilities of being in the open and closed conformations (Figure 3A-C). The wild-type enzyme showed almost equal probability of being in multiple open and closed conformations (Figure 3D). In contrast, the probability distributions for the S45A and A46G enzyme variants were mostly populated by the open conformations of the enzyme (Figure 3D).

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Biochemistry

A

B

Figure 2. RMSD evaluations of PaDADH and the flavin component of FAD for the wild-type, S45A and A46G enzyme variants free in solution. Panels A represent the RMSD of the backbone heavy atoms over time in the three enzymes. Panels B are the RMSD of the flavin N1, N5 and C9 atoms over time in the three enzymes.

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Figure 3. Molecular dynamics analyses of wild-type PaDADH (WT), and the S45A and A46G enzyme variants. Panel A, B and C show the changes in distance over time between the C4 atom of Y53 and the flavin N5 atom in the wild-type (A), S45A (B) and A46G (C) enzyme variants respectively. Panel D shows the probability distributions of the corresponding distance analyses for the three enzymes.

Absorption and Fluorescence Spectroscopy. The S45A and A46G enzyme variants of PaDADH were expressed in E. coli and purified to high level as judged by visual inspection of an SDS-PAGE (data not shown) using the same protocol used for the wild-type enzyme.36 The UV-visible absorption spectra of the mutated enzymes showed the characteristic features of oxidized flavoproteins with absorption maxima in the 370 nm and 450 nm regions of the electro-

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Biochemistry

magnetic spectrum (Figure 4). Both enzyme variants showed small differences in the intensity and maximal wavelength of the low-energy band of the enzyme-bound flavin as compared to the wild-type enzyme (Table 1). In contrast, the high-energy band maximum was red-shifted by 5 nm with 20% higher intensity in the S45A enzyme variant and blue-shifted by 5 nm with 6% higher intensity in the A46G enzyme variant as compared to wild-type PaDADH (Table 1). These changes reflect different protein microenvironments surrounding the flavin in the active site of the two enzyme variants, as expected due to the introduced mutations.

Figure 4. UV-Visible absorption spectra of the S45A (blue), A46G (red) and wild-type PaDADH (black). The spectra were measured in 20 mM sodium phosphate, pH 7.0 and 15 oC. The inset shows the difference absorption spectra of the enzyme bound-flavin in the S45A and A46G enzyme variants minus the wild-type enzyme.

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Table 1. Absorption and Fluorescence Properties of PaDADH. S45A A46G Wild-type Free FAD Maximal absorbance, nm 380, 449 370, 448 375, 446 375, 450 -1 -1 Extinction coefficient, mM cm 10.7, 12.7 9.4, 12.4 8.9, 12.1 9.3, 11.3 Fluorescence emission, nmb 522 ± 1c 522 ± 1 513 ± 1 529 ± 1 Fluorescence intensity 15 ± 1c 25 ± 1 11 ± 1 200 ± 2 UV-visible and fluorescence data were recorded in 20 mM sodium phosphate, pH 7.0 and 15 oC. b Excited at the low energy peak of the flavin. c Standard errors refer to the average of three independent measurements.

Both enzyme variants emitted light with maxima at 522 nm upon excitation of the flavin at the low-energy band (Figure 5) with fluorescence intensities that were 1.5- and 2.3-fold larger than that of wild-type PaDADH (Table 1). The wild-type enzyme, in turn, showed maximal fluorescence emission unusually centered at 513 nm rather than at 529 nm as seen for free FAD in solution, with a relative intensity of ~5% that of free FAD in solution (Table 1).

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Biochemistry

Figure 5. Flavin fluorescence emission spectra of the S45A and A46G enzyme variants compared to the wild-type PaDADH. Spectra were recorded in 20 mM sodium pyrophosphate, pH 7.0 and 15 oC, with enzyme-bound flavin concentrations at 3.5 µM.

kcat/Km and kcat pH-Profiles with D-Arginine. The pH-profiles of the kcat/Km and kcat values with D-arginine as a substrate were determined to establish the effects of the mutations on relevant ionizable groups involved in substrate capture and catalysis. Initially, the app(kcat/Km) and app

kcat values for both the S45A and A46G enzyme variants determined at the extreme pH values

were similar when PMS was kept fixed at 1.0 mM and 1.5 mM (Table 2), consistent with the variant enzymes being fully saturated with 1.0 mM PMS throughout the pH ranges considered. This agrees well with previous results showing that wild-type PaDADH has a Km value ≤10 µM irrespective of whether D-arginine or D-histidine is used as a substrate.27, 28, 36 Consequently, the app

(kcat/Km) and

app

kcat values determined at a fixed concentration of 1 mM PMS with the S45A

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and A46G enzyme variants approximate well the true kcat/Km and kcat values at saturating PMS in the pH range considered.

Table 2. Apparent Steady-State Kinetics of the S45A and A46G Enzyme Variants with DArginine as a Substrate at Fixed Saturating Concentrations of PMS. Enzyme pH [PMS], mM kcat, s-1 kcat / Km, M-1s-1 R2 S45A 5.5 1.0 1.4 ± 0.1 14 ± 1 0.985 1.5 1.5 ± 0.1 11 ± 1 0.983

A46G

10.0

1.0 1.5

290 ± 10 300 ± 10

(2.7 ± 0.2) x 105 (3.1 ± 0.3) x 105

0.996 0.993

6.5

1.0 1.5

23 ± 1 23 ± 1

590 ± 20 600 ± 30

0.998 0.998

1.0 210 ± 10 (3.4 ± 0.3) x 105 0.997 1.5 210 ± 10 (3.1 ± 0.3) x 105 0.995 o In 20 mM sodium pyrophosphate or piperazine at 25 C. Data were acquired at varying concentrations of D-arginine and fixed PMS concentrations of 1.0 and 1.5 mM yielding overlapping saturation curves. 10.0

As shown in Figure 6, the kcat/Km value for both enzyme variants increased with increasing pH values, reaching limiting values at high pH and displaying the requirement for two unprotonated groups with similar pKa value for the enzyme-substrate complex to proceed to catalysis1. This pattern conforms to the results previously reported for the wild-type enzyme with Darginine as a substrate.27

1

With the S45A enzyme variant, it appears that the data points in the region of the pKa value in the log(kcat/Km) pHprofile suggest the presence of a hollow (Figure 6), as for the case of the pH-profiles for the log(kcat). However, the data could not be fit with eq 4, most likely because the hollow in the pH-profile is not pronounced enough. For this reason, the data were fit with eq 5, as for the case of the log(kcat/Km) pH-profiles for the A46G and wild-type enzymes. The conclusions that are drawn from the data of the log(kcat) pH-profile (see Discussion) apply irrespective of whether the log(kcat/Km) pH-profile has a hollow or not.

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Figure 6. pH-Dependence of the kcat/Km and kcat values for the S45A and A46G enzyme variants with D-arginine as a substrate. Enzyme activity was measured at varying concentrations of Darginine and fixed 1 mM PMS at 25 oC. The kcat/Km data were fit to eq 3. With both enzymes, the kcat data were fit better with eq 4 (solid line) than with eq 5 (dotted line).

The log(kcat) value for the variant enzymes also increased with increasing pH values, reaching limiting values at high pH and displaying a hollow with three pH-dependent terms (Figure 6). Hollowed pH-profiles were previously reported with D-lysine as a substrate for wildtype PaDADH and with D-arginine as a substrate for the H48F and E87L enzyme variants of PaDADH.27

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Table 3 summarizes the pKa values and the pH-independent limiting values of the steadystate kinetic parameters for the S45A and A46G enzyme variants determined with D-arginine.

Table 3. Effect of pH on the Steady-State Kinetics with D-Arginine as a Substrate. kcat/Km kcat a -1 -1 Enzymes pKa1 pKa2 (kcat/Km)H (M s ) pKa pKα pKe

(kcat)Ha (s-1)

S45A

7.8b,c

7.8b,c

(2.0 ± 0.6) x 105

6.2 ± 0.3d,e

7.1 ± 0.3d 8.4 ± 0.2d

200 ± 30

A46G

8.0b,c

8.0b,c

(3.5 ± 0.5) x 105

6.3d,e

7.3 ± 0.3d 8.1 ± 0.2d

230 ± 20

6.3b

-

160 ± 20

Wild-typef 7.2b 7.2b (2.6 ± 0.5) x 106 a pH-independent limiting value at high pH b (±0.1) c Determined using eq. 3 d Determined using eq. 4 e βpKa f Taken from reference 27

-

Reductive Half-Reaction. The reductive-half-reaction catalyzed by the A46G and S45A enzyme variants of PaDADH was investigated to obtain insights on the effects of the mutations on substrate binding and flavin reduction. As for the case of the wild-type enzyme,39 D-leucine had to be used as an alternate substrate because with D-arginine flavin reduction was too fast and occurred almost entirely in the mixing time of the stopped-flow instrument (i.e., 2.2 ms) (data not shown). The reductive half-reaction of the S45A and A46G enzyme variants required anaerobic conditions due to a small but not negligible reactivity of the reduced enzyme with molecular oxygen (data not shown); thus, the reductive half-reaction was studied by monitoring the rate of flavin reduction at 450 nm upon mixing the enzyme with varying concentrations of D-leucine. The stopped-flow traces were fit with a single exponential process and the time-resolved absorption spectroscopy demonstrated that both enzyme variants were reduced with D-leucine directly

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Biochemistry

to the hydroquinone state. As shown in Figure 7, with both enzyme variants the observed rate constant for flavin reduction (kobs) increased hyperbolically with increasing concentration of Dleucine. In contrast to the wild-type enzyme, a plot of the kobs values as a function of the concentration of D-leucine showed that both the S45A and A46G enzyme variants could not be saturated with D-leucine as high as 60 mM (Figure 7C). Consequently, only the kred/Kd values could be determined with both variants, yielding values of 90 ± 1 M-1s-1 for the S45A enzyme and 100 ± 2 M-1s-1 for the A46G enzyme. For comparison, wild-type PaDADH had a kred/Kd value of 14,000 ± 10 M-1 s-1 at pH 10.0, as determined in a previous study.39

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Figure 7. Reductive half-reaction of the S45A and A46G enzyme variants of PaDADH with Dleucine as a substrate at pH 10.0 and 25 oC. Panel A and B show the stopped-flow traces of the absorbance changes at 450 nm of the S45A variant (13 µM) and the A46G variant (14 µM), respectively, with 5 mM (black), 15 mM (blue), 25 mM (red), 40 mM (yellow) and 60 mM (green) leucine. Note the log time scale. The instrument mixing time was 2.2 ms. The traces were fit with eq 1. Panel C shows the observed rate constant for flavin reduction of both enzyme variants as a function of D-leucine concentration compared to the wild-type enzyme. The traces were fit to eq 2. The data for the wild-type enzyme are from reference 39. 24


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DISCUSSION A previous crystallographic study on PaDADH suggested that the residues 45-47 and 5056 on the active site loop L1 could adopt two distinct conformations (Figure 1). The 45-47 peptidyl region, which is not in direct contact with the substrate, is located at the FAD-binding domain; the 50-56 peptidyl region is positioned at the entrance of the active site. In the two peptidyl regions of the active site loop L1, the S45, A46 and Y53 residues adopt major conformational changes corresponding to the open and closed conformations. In the closed conformation seen in the enzyme-product complex, the side chain of Y53 blocks the egress of the product from the active site while the side chain of A46 swings closer to the FAD and the side chain of S45 points away from the FAD (Figure 1). In the open conformation seen in the free enzyme state, Y53 points away from the active site towards the bulk solvent, while at the FAD-binding domain the side chain of A46 swings away from the FAD and the side chain of S45 points closer to the FAD (Figure 1). Thus, one can expect the conformation of the A46 and S45 residues known as the Ser/Ala switch to be coupled to Y53, which acts as a gate to allow substrate access to the active site and product release during catalysis. In this study, molecular dynamics, steady-state and rapid reaction kinetics and mutagenesis approaches were used to characterize the impact of the conformational change of the S45/A46 switch in the catalytic function of PaDADH. In wild-type PaDADH, Y53 in the active site portion of loop L1 comprising residues 5056 rapidly samples multiple conformations with almost equal probability of being in open and closed states. Evidence for this conclusion comes from the molecular dynamics simulations of the wild-type enzyme showing that over 1 µs the distance between the C4 atom of Y53 and the N5 atom of the flavin cofactor rapidly oscillates multiple times between ~6 and ~17 Å (Figure 3A). The closed conformations are defined here as those having a distance ≤7 Å between the C4

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atom of Y53 and the N5 atom of the flavin, based on the crystallographic structure of the wildtype enzyme in complex with the iminoarginine product of the reaction resolved to 1.06 Å resolution (Figure 1). The open conformations are those with distances ≥14 Å, based on the crystallographic structure of the wild-type enzyme devoid of bound ligands (Figure 1). While these distances may seem arbitrary, they fulfil the purpose of defining unequivocal and quantifiable boundaries for the open and closed conformations of loop L1. The molecular dynamics simulations also demonstrate that the probability of the wild-type enzyme to have the active site portion of loop L1 in the open and closed conformations is almost uniformly distributed among multiple states (Figure 3D). Similar stochastic movements of loops granting access to the active site of enzymes have been previously demonstrated with molecular dynamics simulations in cytochrome P450, acetylcholinesterase, aryl-alcohol oxidase and choline oxidase.41-45 Furthermore, a swinging of the phenolic side chain of Y53 is seen in the molecular dynamics simulations (data not shown), consistent with the open and closed conformations previously observed in the conformationally averaged crystallographic structure of the enzyme obtained at cryogenic temperature.16 The conformation of the S45/A46 switch in loop L1, which is located at the FAD-binding domain of the enzyme, is coupled to the conformations of the Y53 gate in the substrate-binding domain of loop L1. Evidence to support this conclusion comes from the comparison of the molecular dynamics simulations data on the S45A, A46G, and wild-type forms of PaDADH. Distances significantly >7 Å between the C4 atom of Y53 and the N5 atom of the flavin are more probable in the S45A and A46G enzyme variants, consistent with an increased probability of loop L1 to be in open conformations; in contrast, the probability to be in open and closed conformations is almost equally distributed in the wild-type enzyme (Figure 3). Thus, the replace-

26


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Biochemistry

ment of S45 or A46 with smaller residues shifts the probability distribution of the Y53 gate toward open conformations. This, in turn, is consistent with the two enzyme variants having active sites that are more exposed to the bulk solvent than the wild-type enzyme. In agreement with a more solvent-exposed flavin in the enzyme variants, a 1.5- to 2.3-fold increase in fluorescence intensity of the enzyme-bound flavin is observed in the S45A and A46G enzymes variants with respect to the wild-type enzyme (Table 1).46, 47 The 9 nm bathochromic shift of the maximal wavelength of the emission band from 513 nm to 522 nm in the S45A and A46G enzyme variants compared to that of the wild-type enzyme is further consistent with an increase in hydrophilicity that is consequent to the flavin being more solvent-exposed in the enzyme variants2 (Table 1).46-48 An increased probability of the active site gate Y53 to be in open conformations decreases the likelihood of efficiently capturing the physiological substrate D-arginine in the enzymesubstrate complex that proceeds to catalysis. Evidence to support this conclusion comes from the pH-profiles of the kcat/Km value with D-arginine as substrate, showing a 11- to 13-fold decrease in the pH-independent kcat/Km values for the S45A and A46G enzyme variants as compared to the wild-type enzyme (Table 3). As in the case of wild-type PaDADH, the ascending limbs in the pH-profiles of the log(kcat/Km) value with D-arginine as a substrate for the S45A and A46G enzyme variants showed the presence of two unprotonated groups with indistinguishable pKa values, which were previously assigned to the α-NH3+ group of the D-arginine substrate and the

2

It is noteworthy that the wild-type enzyme has maximal fluorescence emission at 513 nm rather than 529 nm as for free FAD in solution (Table 3), with a relative intensity of ~5% that of free FAD. Typically, while the intensity of fluorescence emission of enzyme-bound flavins can be significantly lower than free FAD due to quenching by the 55-57 protein microenvironment surrounding the flavin, the emission maxima are in the range from 525 to 535 nm. The unusual behavior of wild-type PaDADH with an emission maximum at 513 nm is consistent with the crystallographic structure of the enzyme showing that the flavin is buried inside the FAD-binding domain of the enzyme with little exposure to the bulk solvent.

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side chain of E87 interacting with the guanido group of the substrate.27 In that same study Darginine was established as a sticky substrate for the wild-type form of PaDADH, based on an outward shift of the apparent pKa value from an intrinsic value of ~9.5 to 7.2 determined in the pH-profile of the kcat/Km value with D-arginine.27 In this respect, the comparison of the apparent pKa values of 7.8-8.0 determined in this study for the S45A and A46G enzyme variants with that of 7.2 determined for wild-type PaDADH demonstrates that D-arginine is less sticky in the two enzyme variants as compared to the wild-type enzyme. This is likely due to the active site portion of loop L1 having increased probability to be in open conformations in the S45A and A46G enzyme variants than in the wild-type enzyme, which would favor substrate dissociation from the enzyme-substrate complex rather than proceeding through catalysis (Figure 3). Substrate release from the enzyme-substrate complex is even more exacerbated with the alternate substrate Dleucine, which lacks the anchoring guanido group present on the side chain of D-arginine. Indeed, the second-order rate constant kred/Kd determined in the reductive half-reaction with the alternate substrate D-leucine is ~150-fold lower in the S45A and A46G enzyme variants than in the wild-type form of PaDADH. The dynamics of loop L1 plays an important role for the optimal orientation of the Darginine substrate in the enzyme-substrate complex that proceeds to catalysis. This conclusion is supported by the presence of a hollow on the acidic limbs of the pH-profiles of the log(kcat) value of the S45A and A46G enzymes with D-arginine as a substrate (Figure 6). Hollowed pH-profiles are typically seen in enzymatic reactions involving sticky substrates with a high propensity to proceeding to catalysis rather than dissociating from the enzyme-substrate complex, and slow exchange of protons in the catalytic steps (i.e., sticky protons).40, 49-51 A previous study on the wild-type form of PaDADH showed a hollowed pH-profile of the log(kcat) value with the alter-

28


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Biochemistry

nate substrate D-lysine, but not with the physiological substrate D-arginine.27 It was concluded that D-lysine has a different orientation within the enzyme-substrate complex undergoing catalysis than D-arginine, preventing a fast and unrestricted release of the sticky proton from its αNH3+ group in the catalytic step.27 Thus, the replacement of S45 and A46 with smaller amino acid residues results in the restricted release of the proton from the α-NH3+ group of the physiological substrate D-arginine, like previously seen in the wild-type enzyme with the alternate substrate D-lysine. This is illustrated in Scheme 2, in which the α-amino group of D-arginine is either protonated or unprotonated before binding to form the enzyme-substrate complex that proceeds to catalysis in the S45A and A46G enzyme variants, with the deprotonation of the PaDADHox-SH+ (i.e., step k15/k16) occurring on a timescale comparable to that of the subsequent catalytic step of flavin reduction (i.e., k3). A similar slow equilibration of PaDADHox-SH+ and PaDADHox-S with D-arginine was recently observed in the case of the H48F variant of PaDADH.27 Interestingly, H48 belongs to the same Loop L1 that harbors S45 and A46, suggesting that a common mechanism may be responsible for the restricted proton movements in the H48F, S45A and A46G enzyme variants. In the X-ray crystallographic structure of the wild-type enzyme in complex with the iminoarginine product of reaction two water molecules bridge the histydyl N1 atom of H48 with the α-imino group of the ligand (Figure 8). Thus, it is possible that the replacement of S45 or A46 misplaces the H48 residue and the associated water network in the active site of the enzyme with consequent misplacement of the substrate in the enzymesubstrate complex that proceeds to catalysis.

Scheme 2: Reductive Half-Reaction of the S45A and A46G variants of PaDADHa.

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a

In the wild-type enzyme both the top and bottom paths are relevant with D-lysine, but only the

top path is relevant with D-arginine as a substrate.27

Figure 8. Position of the S45 and A46 residues in wild-type PaDADH. Loop L1 is colored in cyan where H48 residue is forming a hydrogen bond with the reaction product (IAR) through two water molecules. FAD is represented by its isoalloxazine ring with the C atoms colored in yellow.

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Biochemistry

Based on a sub-optimal orientation of the D-arginine substrate in the enzyme-substrate complex that proceeds to catalysis, one would expect altered rate constants for flavin reduction upon replacing S45 or A46 in PaDADH. However, with both the S45A and A46G enzyme variants flavin reduction with D-arginine occurred predominantly in the mixing time of the stoppedflow spectrophotometer (data not shown), as previously shown also for the wild-type form of the enzyme.36 Thus, a direct comparison of the rate constants for flavin reduction with the physiological substrate could not be carried out. When the alternate, slow substrate D-leucine was used with the S45A and A46G enzymes no saturation could be achieved with substrate concentrations as high as 60 mM (Figure 7). In contrast, the wild-type form of PaDADH was previously shown to have a Kd value of 6.8 mM and a kred value of 91 s-1 with D-leucine as a reducing substrate under the same conditions, i.e., pH 10 and 25 oC.39 These data taken together do not allow drawing conclusions on how the replacement of S45 or A46 may impact the catalytic step of flavin reduction. In summary, we have used molecular dynamics, steady-state and rapid reaction kinetics, pH effects, fluorescence spectroscopy and site-directed mutagenesis approaches to elucidate the dynamics of the active site loop L1 and their impact on substrate capture and catalysis in PaDADH. The molecular dynamics simulations complement previous crystallographic data on the wild-type form of PaDADH showing that Y53 in the active site portion of loop L1 fluctuates between open and closed conformations and acts as a gate to control the access of the active site.

16

In loop L1, the movement of the S45/A46 switch, which is not in direct contact with the substrate, is coupled with the open and closed conformations of the gate Y53 in the active site of the enzyme. Indeed, the replacement of S45 to alanine or A46 to glycine through site-directed mutagenesis yielded enzyme variants in which the gate has increased probabilities to be in open con-

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formations. In this respect, loop L1 in PaDADH has similar behavior compared to the Ω-loops observed in triosephosphate isomerase and phosphoenolpyruvate carboxykinase, where point mutations in those loops increased the probability of the loop-open conformations.52, 53 The increased probability of the active site loop L1 in the S45A and A46G enzyme variants of PaDADH to be in the open conformations impaired the capture of the physiological substrate, Darginine. Furthermore, the two enzyme variants were unable to orient properly the α-NH3+ group of D-arginine in the enzyme-substrate complex that proceed to catalysis. In contrast to wild-type PaDADH, the two enzyme variants could not be saturated with the alternate reducing substrate D-leucine in the reductive half-reaction, most likely due to an active site that is more open to the bulk solvent. Mutations that shifted the probability of the active site loop toward a more opened conformation and thereby affect negatively the accommodation of the substrate in the active site of the enzyme and subsequent catalysis had been reported among well-characterized enzymes including triosephosphate isomerase, dihydrofolate reductase and phosphoenolpyruvate carboxykinase.6, 52-54 This study shows the importance of loop dynamics for substrate capture and catalysis and, most importantly, its alterations through mutagenesis of a portion, which is not in direct contact with the ligand in the active site of the enzyme, have been demonstrated through experimental and computational approaches in a flavin-dependent enzyme.

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AUTHOR INFORMATION Corresponding Author * Department of Chemistry, Georgia State University, P.O. Box 3965, Atlanta, GA 30302-3965; Phone: (404) 413-5537, Fax: (404) 413-5505, Email: [email protected] Funding This work was supported in part by National Science Foundation Grant CHE-1506518 (G.G.), National Science Foundation Grant MCB-1517617 (D.H.) and the Georgia Research Alliance. M.S. was supported by the U.S. Department of Education GAANN Fellowship and is a Georgia State University Bio-Bus Fellow. Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS The authors thank the participants of the Atlanta Flavin Meeting: Dale Edmondson, Andreas Bommarius and Stefan Lutz and their research groups for insightful suggestions. Daniel Ouedraogo also thanks Susan and Allen Scott, and Cheryl and Steve Shuler for their kind and generous support.

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Biochemistry

Table 1. Absorption and Fluorescence Properties of PaDADH. S45A A46G Wild-type Free FAD Maximal absorbance, nm 380, 449 370, 448 375, 446 375, 450 -1 -1 Extinction coefficient, mM cm 10.7, 12.7 9.4, 12.4 8.9, 12.1 9.3, 11.3 Fluorescence emission, nmb 522 ± 1c 522 ± 1 513 ± 1 529 ± 1 Fluorescence intensity 15 ± 1c 25 ± 1 11 ± 1 200 ± 2 UV-visible and fluorescence data were recorded in 20 mM sodium phosphate, pH 7.0 and 15 oC. b Excited at the low energy peak of the flavin. c Standard errors refer to the average of three independent measurements.

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Table 2. Apparent Steady-State Kinetics of the S45A and A46G Enzyme Variants with DArginine as a Substrate at Fixed Saturating Concentrations of PMS. Enzyme pH [PMS], mM kcat, s-1 kcat / Km, M-1s-1 R2 S45A 5.5 1.0 1.4 ± 0.1 14 ± 1 0.985 1.5 1.5 ± 0.1 11 ± 1 0.983

A46G

10.0

1.0 1.5

290 ± 10 300 ± 10

(2.7 ± 0.2) x 105 (3.1 ± 0.3) x 105

0.996 0.993

6.5

1.0 1.5

23 ± 1 23 ± 1

590 ± 20 600 ± 30

0.998 0.998

1.0 210 ± 10 (3.4 ± 0.3) x 105 0.997 1.5 210 ± 10 (3.1 ± 0.3) x 105 0.995 o In 20 mM sodium pyrophosphate or piperazine at 25 C. Data were acquired at varying concentrations of D-arginine and fixed PMS concentrations of 1.0 and 1.5 mM yielding overlapping saturation curves. 10.0

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Biochemistry

Table 3. Effect of pH on the Steady-State Kinetics with D-Arginine as a Substrate. kcat/Km kcat a -1 -1 Enzymes pKa1 pKa2 (kcat/Km)H (M s ) pKa pKα pKe

(kcat)Ha (s-1)

S45A

7.8b,c

7.8b,c

(2.0 ± 0.6) x 105

6.2 ± 0.3d,e

7.1 ± 0.3d 8.4 ± 0.2d

200 ± 30

A46G

8.0b,c

8.0b,c

(3.5 ± 0.5) x 105

6.3d,e

7.3 ± 0.3d 8.1 ± 0.2d

230 ± 20

-

160 ± 20

f

b

b

6

Wild-type 7.2 7.2 (2.6 ± 0.5) x 10 pH-independent limiting value at high pH b (±0.1) c Determined using eq. 3 d Determined using eq. 4 e βpKa f Taken from reference 27

b

6.3

a

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Scheme 1: D-Arginine Oxidation by PaDADH

Scheme 2: Reductive Half-Reaction of the S45A and A46G variants of PaDADHa.

a

In the wild-type enzyme both the top and bottom paths are relevant with D-lysine, but only the

top path is relevant with D-arginine as a substrate.27

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Biochemistry

Figure 1. Open and closed conformations of loop L1 in wild-type PaDADH (PDB: 3NYC). Panels A and B show the overall structure of the enzyme with the flavin-binding and substratebinding domains and loop L1 in the open (orange) and closed (green) conformations. IAR is the iminoarginine product bound in the active site. Panel C shows the distances between the flavin N5 atom and the C4 atom of Y53, and between the αC atom of Y53 in the open and closed conformations. Note that in the closed conformation there is a hydrogen bond interaction between the hydroxyl O atom of S45 and the 2’-OH of the ribityl moiety of the FAD cofactor.

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A

B

Figure 2. RMSD evaluations of PaDADH and the flavin component of FAD for the wild-type, S45A and A46G enzyme variants free in solution. Panels A represent the RMSD of the backbone heavy atoms over time in the three enzymes. Panels B are the RMSD of the flavin N1, N5 and C9 atoms over time in the three enzymes.

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Biochemistry

Figure 3. Molecular dynamics analyses of wild-type PaDADH (WT), and the S45A and A46G enzyme variants. Panel A, B and C show the changes in distance over time between the C4 atom of Y53 and the flavin N5 atom in the wild-type (A), S45A (B) and A46G (C) enzyme variants respectively. Panel D shows the probability distributions of the corresponding distance analyses for the three enzymes.

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. UV-Visible absorption spectra of the S45A (blue), A46G (red) and wild-type PaDADH (black). The spectra were measured in 20 mM sodium phosphate, pH 7.0 and 15 oC. The inset shows the difference absorption spectra of the enzyme bound-flavin in the S45A and A46G enzyme variants minus the wild-type enzyme.

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Biochemistry

Figure 5. Flavin fluorescence emission spectra of the S45A and A46G enzyme variants compared to the wild-type PaDADH. Spectra were recorded in 20 mM sodium pyrophosphate, pH 7.0 and 15 oC, with enzyme-bound flavin concentrations at 3.5 µM.

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Figure 6. pH-Dependence of the kcat/Km and kcat values for the S45A and A46G enzyme variants with D-arginine as a substrate. Enzyme activity was measured at varying concentrations of Darginine and fixed 1 mM PMS at 25 oC. The kcat/Km data were fit to eq 3. With both enzymes, the kcat data were fit better with eq 4 (solid line) than with eq 5 (dotted line).

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Biochemistry

Figure 7. Reductive half-reaction of the S45A and A46G enzyme variants of PaDADH with Dleucine as a substrate at pH 10.0 and 25 oC. Panel A and B show the stopped-flow traces of the absorbance changes at 450 nm of the S45A variant (13 µM) and the A46G variant (14 µM), respectively, with 5 mM (black), 15 mm (blue), 25 mM (red), 40 mM (yellow) and 60 mM (green) leucine. Note the log time scale. The instrument mixing time was 2.2 ms. The traces were fit with eq 1. Panel C shows the observed rate constant for flavin reduction of both enzyme variants as a function of D-leucine concentration compared to the wild-type enzyme. The traces were fit to eq 2. The data for the wild-type enzyme are from reference 39. 49


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Position of the S45 and A46 residues in wild-type PaDADH. Loop L1 is colored in cyan where H48 residue is forming a hydrogen bond with the reaction product (IAR) through two water molecules. FAD is represented by its isoalloxazine ring with the C atoms colored in yellow.

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Biochemistry

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Importance of Loop L1 Dynamics for Substrate Capture and Catalysis

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