8840
Biochemistry 2008, 47, 8840–8854
Catalytic Mechanism and Substrate Specificity of the β-Subunit of the Voltage-Gated Potassium Channel† Srinivas M. Tipparaju, Oleg A. Barski, Sanjay Srivastava, and Aruni Bhatnagar* DiVision of Cardiology, Department of Medicine, Institute of Molecular Cardiology, UniVersity of LouisVille, LouisVille, Kentucky 40202 ReceiVed February 21, 2008; ReVised Manuscript ReceiVed June 10, 2008
ABSTRACT: The β-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvβ proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvβ2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min-1) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvβ2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvβ proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.
Most eukaryotic cells express a diverse range of voltagegated potassium (Kv)1 channels (1). In excitable cells, such as nerves and muscles, Kv channels regulate the generation and duration of action potential, firing patterns, pacemaking, and neurotransmitter release (2–4). In nonexcitable cells, these channels participate in volume regulation, hormonal secretion, proliferation, and apoptosis (5–8). Native mammalian Kv channels are large macromolecular complexes composed of four pore-forming transmembrane proteins (Kv R-subunits) that regulate K+ efflux. The cytosolic domains of the Kv1 and -4 R-subunits associate with auxiliary β-subunits that regulate channel assembly and function. In mammals, three distinct Kvβ genes have been identified. These genes encode proteins with a highly conserved C-terminus and a variable N-terminal domain. The conserved C-terminus of Kvβ proteins folds into an (R/β)8 or triosephosphate isomerase (TIM) barrel motif, which is the most common structural scaffolding among enzymes involved in † This work was partly supported by NIH Grants HL-544771, HL59378, and ES-11860 (to A.B.) and HL-089372 (to O.A.B.) and a fellowship from the American Heart Association (0425439B, to S.M.T.). * To whom correspondence should be addressed: Division of Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202. Telephone: (502) 852-5966. Fax: (502) 852-3663. E-mail:
[email protected]. 1 Abbreviations: 4-NB, 4-nitrobenzaldehyde; 4-NAP, 4-nitroacetophenone; 4-HNE, 4-hydroxy-trans-2-nonenal; DHN, 1,4-dihydroxytrans-2-nonene; 4-ONE, 4-oxo-trans-2-nonenal; Kv, voltage-gated potassium; Kvβ, voltage-gated potassium channel β-subunit; AKR, aldoketo reductase; SEM, standard error of the mean.
metabolism and biosynthesis (9). The TIM barrel also forms an efficient fold for high-affinity binding of flavin and pyridine nucleotides that participate in catalyzing oxidationreduction reactions (9). The TIM barrel structure of Kvβ proteins bears a strong structural resemblance and sequence similarity to proteins of the aldo-keto reductase (AKR) superfamily, and the catalytic features of the AKR active site are conserved in the C-terminal domain of Kvβ proteins (10–12). On the basis of this homology, the Kvβ proteins have been classified as a distinct family (AKR6) within the AKR superfamily of proteins (13). Our previous studies show that like other AKRs, the Kvβ proteins bind to pyridine nucleotides with high affinity (14, 15). We have found that binding of reduced nucleotides supports N-terminally mediated inactivation of KvR currents by Kvβ, whereas oxidized nucleotides remove inactivation of K+ currents generated by Kv Rβ-subunits (16). These observations support the notion that the β-subunits impart metabolic sensitivity to Kv currents. Such coupling might allow the Kv channel to sense changes in cell metabolism and oxygenation (17). Nevertheless, it remains unclear whether binding of pyridine nucleotide to Kvβ proteins by itself is required for the regulation of Kv activity or if catalysis is necessary to change the redox state of the nucleotide bound to Kvβ. Clearly, further studies on the catalytic activity and substrate specificity of Kvβ are required to understand how it regulates Kv function.
10.1021/bi800301b CCC: $40.75 2008 American Chemical Society Published on Web 08/02/2008
Catalytic Mechanism of Kvβ Several lines of evidence suggest that Kvβ proteins are catalytically active and exhibit AKR properties. The AKR active site is conserved in Kvβ proteins, and the catalytic residues are positioned for efficient carbonyl binding. Moreover, mutations at the active site markedly affect the inactivating properties of Kvβ (15, 18, 19). Indeed, weak catalytic activity of Kvβ2 with aromatic aldehydes has been reported previously (18). Nevertheless, the catalytic and kinetic mechanisms of the protein remain poorly understood, and its substrate specificity is unknown. Specifically, it is not clear whether the protein displays cooperativity, which steps limit the overall catalytic rate, and which structural features facilitate substrate binding to the active site. This study was therefore designed to identify the reaction sequence, to determine the kinetic mechanism of catalysis, and to examine the substrate specificity of the protein. Our results show that Kvβ follows a rapid equilibrium bi-bi ordered reaction mechanism. These properties may be important aspects of Kvβ function. EXPERIMENTAL PROCEDURES Mutagenesis and Expression of KVβ2. The cDNA encoding rat Kvβ2 (rKvβ2) was provided by M. Li (Johns Hopkins University, Baltimore, MD). The construct for bacterial expression of rKvβ2 (NP_059000) with a His tag at its N-terminus in the pET28a vector was generated as described previously (14). Site-directed mutagenesis was performed directly in a pET28a vector using a QuikChange XL sitedirected mutagenesis kit (Stratagene). The sequences of the PCR primers were 5′-CGATACGGCGGAGGTCTTCGCAGCTGGCAAGGCTG-3′ (forward) and 5′-CAGCCTTGCCAGCTGCGAAGACCTCCGCCGTATCG-3′ (reverse). The complete sequence of the mutated Kvβ2 insert was confirmed by DNA sequencing. Wild-type and mutant Kvβ2 were expressed in the BL-21(DE3) strain of Escherichia coli and purified to homogeneity as described previously (14). Protein Purification. The C-terminal domain of Kvβ2 (amino acids 39-367) was expressed with a His tag at its N-terminus in the BL-21 strain of E. coli. The bacterial pellet was lysed in ice-cold binding buffer containing 150 mM NaCl, 20 mM Tris, and 5 mM imidazole (pH 7.9). The supernatant was incubated with Ni-affinity agarose beads (Qiagen) for 2-3 h at 4 °C. The Ni column was washed with binding buffer with 10 times the bed volume, and the protein was eluted with imidazole. A small volume from each eluted fraction (2 µL) was separated by SDS-PAGE. The purified proteins were stored at 4 °C in 0.2 M potassium phosphate buffer (pH 7.4) and used within 4-6 weeks. The concentration of the protein was measured using Bradford’s assay (20). NADPH Binding Studies. Binding of NADPH to WT or Y90F Kvβ2 was assessed by fluorescence titration as described previously (14). Briefly, the protein (15 µg) was equilibrated in 2 mL of 0.2 M potassium phosphate buffer at room temperature for 10 min, and changes in fluorescence were measured using an excitation wavelength of 280 nm and emission at 340 nm. Aliquots of NADPH were added, and changes in emission were recorded. Fluorescence changes obtained by titrating tryptophan with NADPH were used to correct for the inner filter effect (14). Dissociation constants were calculated using the following equation:
Biochemistry, Vol. 47, No. 34, 2008 8841 ∆F ) ∆FmaxNADPH ⁄ (Kd + NADPH)
(1)
ActiVity Measurements in Multiple- and Single-TurnoVer Mode. Activity assays were performed in multiple- or singleturnover mode (18, 21). Multiple turnovers were assessed in quartz microcuvettes containing, in a volume of 250 µL, Kvβ2 (15-20 µM), NADPH (150 µM), and 0.2 M phosphate (pH 7.4) incubated at 37 °C. The reaction was started by adding the substrate. Loss of NADPH was monitored by following changes in absorbance at 340 nm for 15 min on a spectrophotometer (Cary Varian 50). For the measurement of single-turnover activity, the Kvβ2 protein was preequilibrated at 37 °C and the reaction was initiated by adding the substrate (0.2-2 mM). The reaction was monitored continuously by measuring changes in A360 for 30-60 min at 37 °C to follow the oxidation of NADPH bound to Kvβ2. Synthesis of Deuterated NADPH. NADPH and NADPD were synthesized by using alcohol dehydrogenase from Thermoanaerobium brockii (Sigma Chemical Co.) as described previously (22). For synthesis, alcohol dehydrogenase 2U (1.21 mg) was incubated with 2 mM NADP+ in 50 mM Tris-HCl (pH 7.8) and 300 mM 2-propanol or 2-propanold8 (Sigma Chemical Co.) for 1 h at 40 °C. The progress of the reaction was monitored by measuring changes in absorbance at 340 nm. After completion, the reaction mixture was loaded on an anion-exchange column pre-equilibrated with 20 mM Tris-HCl (pH 9.0). The column was washed with 25-30 mL of 20 mM Tris-HCl (pH 9.0), and the reduced cofactor NADPH/D was eluted by using 250 mM NaCl and 50 mM Tris (pH 7.8). The absorbance of the eluted fractions at 260 and 340 nm was recorded. Fractions with an A260/ A340 ratio of >2.4 were pooled, and the pH of the mixture was adjusted to 9.0 with NaOH. Synthesized pyridine nucleotides were stored at -80 °C until further use. Nucleotide Replacement Experiments. The Kvβ2 protein, purified from bacteria, remains tightly bound to NADPH, and the cofactor is not removed by dialysis. Hence, for kinetic isotope effect studies, NADPH bound to Kvβ was removed by oxidizing it to NADP+, which dissociates from the protein more readily than NADPH. For this, Kvβ2, purified from bacteria, was incubated with 4-nitrobenzaldehyde (600 µM), and the disappearance of NADPH was monitored at 360 nm. After the reaction had been driven to completion, the reaction mixture was transferred to a dialysis cassette (10000 Da cutoff, Pierce) and dialyzed against 0.2 M potassium phosphate (pH 7.4) at 4 °C for 16-20 h. The protein was then incubated with reagent NADPH or NADPD for 30 min at 4 °C. Excess coenzyme was removed by equilibrium dialysis. The progress of the reaction was monitored at each step by measuring changes in absorbance at 360 nm. Kinetic Isotope Effects. To study kinetic isotope effects, 150 µM synthesized NADPH or NADPD was mixed with 20 µM Kvβ2 in 0.2 M potassium phosphate (pH 7.4) at 37 °C. The enzyme reaction was started by adding varying concentrations (0.1-2 mM) of the substrate. Coenzyme oxidation was monitored at 340 nm for 15 min at 37 °C. For the calculation of velocity, only the initial, linear part of the reaction was used. Kinetic isotope effects on singleturnover reactions were calculated from the rate of oxidation of NADPH or NADPD bound to Kvβ2 measured at 37 °C for 30 min after the addition of the substrate.
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Biochemistry, Vol. 47, No. 34, 2008
Product Identification. Reduction of phospholipid aldehyde 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) to 1-palmitoyl-2(5)-hydroxyvaleroyl-sn-glycero-3-phosphorylcholine (PHVPC) by Kvβ2 was followed by electrospray mass spectrometry (ESI+/MS) as described previously (23). The reaction mixture containing 35 µM Kvβ2, 150 µM NADPH, and 80 µM POVPC was incubated at 37 °C for 3 h. Additional NADPH was added to the reaction mixture at 45 min intervals. The reaction was stopped after 3 h, and the phospholipid in the mixture was extracted by using the Bligh and Dryer (24) procedure. For ESI+/MS analysis, a MicroMass ZMD 2000 mass spectrophotometer (Waters-Micromass, Milford, MA) was used. The injection solvent was a 2:1 (v/v) methanol/chloroform mixture containing 1.0% acetic acid in positive ionization mode and a 2:1 (v/v) methanol/chloroform mixture containing 10 mM ammonium hydroxide in negative ionization mode. Samples were injected into the spectrometer using a Harvard syringe pump at a flow rate of 25 µL/min. The ESI+/ MS operating parameters were as follows: capillary voltage, 3.38 kV; cone voltage, 25 V; extractor voltage, 9 V; RF lens voltage, 0.9 V; and source block and desolvation temperatures, 100 and 200 °C, respectively. Nitrogen was used as the nebulizer gas at a flow rate of 3.4 L/h. Spectra were acquired at a rate of 275 atomic mass units per second over the mass range of 2-1000 atomic mass units and were averaged over a period of 5 min or 100 scans (23). HPLC Analysis. Reagent 4-hydroxy-trans-2-nonenal (4HNE) and 1,4-dihydroxynonenal (DHN) were synthesized as described previously (25). The 4-oxo-2-nonenol was synthesized by reducing 4-oxo-2-nonenal (4-ONE) with aldose reductase (25). Reagent 4-HNE, 4-ONE, DHN, and 4-oxo-2-nonenol were separated by HPLC using a Varian reverse phase ODS C18 column pre-equilibrated with 0.1% aqueous trifluoroacetic acid. The compounds were eluted using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. The gradient was established such that the level of solvent B reached 26% in 30 min and was held at this value for 10 min. Over the next 10 min, the level of solvent B reached 60%. After an additional 5 min, it reached 100% and was held at this value for 10 min. Under these conditions, DHN, 4-oxo-2-nonenol, 4-HNE, and 4-ONE eluted with retention times of 31, 37, 43, and 53 min, respectively. Analysis of the Reduced Product by Gas ChromatographyMass Spectrometry (GC-MS). HPLC-purified peak II was extracted in dichloromethane and dried under nitrogen. The samples were derivatized with 50 µL of BSTFA-TMS [N,Obis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane] for 5 min at 80 °C. An aliquot (1 µL) of the mixture was utilized for gas chromatography-electron impact ionization mass spectrometry (GC-EI-MS) analysis. GC-EI-MS analysis was performed using an HP-6890/HP5973 GS/MS system (Hewlett-Packard, Palo Alto, CA). Compounds were separated on a bonded phase capillary column (DB-5MS, 30 m × 0.25 mm × 0.25 µm film thickness from J&W Scientific, Folson, CA). The GC injection port and interface temperature were set at 280 °C. Injections were made in the splitless mode with the inlet port purged for 1 min following injection. The GC oven temperature was held initially at 100 °C for 1 min, then increased at a rate of 10 °C/min to
Tipparaju et al. 280 °C, and then held there for 5 min. Under these conditions, the retention time of 4-oxo-2-nonenol was 8.06 min. Hammett Plot Analysis. The kinetic constants, Km and kcat, were obtained with the series of benzaldehyde compounds. For each para-substituted benzaldehyde, values for electronic (σ+), steric (υ), and hydrophobic (Π) parameters were obtained from published literature (26–28). The log kcat, log Km, or log kcat/Km values were plotted versus σ+, and the correlation coefficients were obtained from multivariate weighted analysis as described previously (22, 29) for the following equation using SAS 9.1: log kcat ⁄Km′ log kcat, or log Km ) Fσ+ + Aυ + BΠ + C (2) where σ+ is the Hammett constant for electronic effects, Π is the Hansch constant for the hydrophobic interactions, and υ is the steric constant. F, A, B, and C are parameter coefficients. Data Analysis. Steady-state kinetic constants kcat and Km were calculated by fitting the Michaelis-Menten equation directly in the hyperbolic form to the initial velocities using the Marquardt-Levenberg algorithm. For nonlinear regression analysis, SigmaPlot 9.0 was used. Single-turnover traces were analyzed using a single-exponential decay equation: A360 ) ae-ktranst + b
(3)
Calculated pseudo-first-order rate constants, ktrans, were then plotted against substrate concentration and analyzed using nonlinear regression analysis. Kinetic isotope effects were calculated from the initial velocities obtained with NADPH and NADPD at various substrate concentrations using the following equations: V ) VMA ⁄ [Km(1 + FiEkcat ⁄Km) + A(1 + FiEkcat)]
(4)
V ) VMA ⁄ [(Km + A)(1 + FiE)]
(5)
where A is the concentration of aldehyde substrate, Fi is the fraction of deuterium in the NADPH/NADPD cofactor, and Ekcat/Km and Ekcat are the isotope effects minus 1 on kcat/Km and kcat, respectively. Equation 5 assumes that Ekcat/Km ) Ekcat. Multivariate regression analysis was performed using SigmaPlot 9.0. Data are presented as means ( SEM. Intergroup comparisons were made using the unpaired Student’s t-test, and a P of 7.5 or
