Subscriber access provided by University of Newcastle, Australia
Article
Significance of individual residues at the regulatory site of yeast pyruvate decarboxylase for allosteric substrate activation Michael Spinka, Sebastian Seiferheld, Philipp Zimmermann, Elena Bergner, Anne-Kathrin Blume, Angelika Schierhorn, Tom Reichenbach, Robert Pertermann, Christiane Ehrt, and Stephan König Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01158 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 51
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
Biochemistry
Significance of individual residues at the regulatory site of yeast pyruvate decarboxylase for allosteric substrate activation Michael Spinka, Sebastian Seiferheld, Philipp Zimmermann, Elena Bergner, Anne-Kathrin Blume¶, Angelika Schierhorn, Tom Reichenbach§, Robert Pertermann, Christiane Ehrt&, and Stephan König* Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg, Halle (Saale) KEYWORDS steady state and transient kinetics, in vitro mutagenesis, protein purification, small-angle X-ray scattering (SAXS), stopped-flow, microscopic constants, thiamine diphosphate, regulatory dyad ABBREVIATIONS ScPDC, pyruvate decarboxylase from Saccharomyces cerevisiae.
ACS Paragon Plus Environment
1
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
Page 2 of 51
ABSTRACT The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated the variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the activate state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221 ensuring a rapid nucleophilic attack of the thiolate sulphur on the C2 atom of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly diminished for A287G and significantly retarded for S311A.
ACS Paragon Plus Environment
2
Page 3 of 51
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
Biochemistry
The allosteric regulation of catalytic activity represents a direct and rapid mechanism to control turnover rates especially at branching points of metabolic pathways. The corresponding allosteric enzymes are typically able to assume several structurally well-defined conformations with distinct regulatory and catalytic features. Domain movements may cause changes of the specific active site architecture resulting in significantly altered catalytic properties. The effectors inducing the conformation changes by binding at regulatory sites may be specific metabolites, substrates or products of the same or functionally related metabolic pathways. In some cases the substrate itself acts as regulator. Pyruvate decarboxylase from Saccharomyces cerevisiae (ScPDC) represents a paradigmatic example of this category of substrate activation. This enzyme species is almost catalytically inactive in the absence of its substrate pyruvate1. Upon addition of the substrate it takes 10-20 seconds to reach the full activity at pyruvate concentrations and temperatures typical for living yeast cells2. Thiamine diphosphate in complex with magnesium ion is the essential cofactor of pyruvate decarboxylase (2-oxo acid carboxy lyase, EC 4.1.1.1). Only for a minority of thiamine diphosphate dependent enzymes an allosteric mechanism has been shown to be operative. Although the architecture of their regulatory sites was characterized in detail by X-ray crystallography, little is known about the specific roles of individual amino acid residues involved in regulation. In the case of ScPDC the pivotal role of cysteine residues for allosteric enzyme activation was postulated since the eighties based on investigations of chemical modifications3, isotope effects4, and proton inventories5. From the analyses of the first crystal structures of the enzyme species and kinetic studies on various cysteine variants, residue C221 was favored as the core of the regulatory site6-9. The intricate question of signal transfer pathway from the regulatory to the active site across a distance of about 20 Å remained a matter of debate over the years10-18.
ACS Paragon Plus Environment
3
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
Page 4 of 51
ScPDC is a homo-oligomer. At protein concentrations above 1mg/mL (16 µM subunit) and pH optimum (around 6) tetramers dominate, otherwise a dimer tetramer equilibrium prevails. Monomers are hardly detectable as an autonomous species19-21. The subunits (61.5 kDa) consist of three α/β-domains4,
10-12, 22
. The middle domain harbors the regulatory site. The cofactor
molecules are bound between the two other domains at the interface of different subunits so that each thiamine diphosphate-magnesium complex links two monomers and thus support the structural integrity of the protein component (figure 1). Consequently, the dimer is the smallest catalytically active unit of ScPDC23-24. We have previously shown that substrate binding at the regulatory site causes the fixation of two, otherwise flexible loop regions (side chains 104-113, and 288-304, respectively) together with the translocation of the C-terminal helix thus shielding the active site against the solvent and leading to a remarkable increase of the catalytic activity of the enzyme11-12, 25. This is plausible because a non-polar environment supports the heterolytic cleavage of the C-H bond at the C2 position of the thiazolium moiety of the cofactor and thus increases the stability of the ylide species of thiamine diphosphate26, 27. Jordan and coworkers had postulated a direct interaction of amino acid residues along a signal transfer pathway from C221 to the C2 atom of thiamine diphosphate buttressed mainly by kinetic analyses of a broad range of variants10, 13-14, 16-17, 28-29. By comparing high resolution crystal structure models of yeast decarboxylases, both wild type and variants, crystallized in the presence of substrate and artificial activators, we were able to trace the signal transfer in detail at the structural level25. Moreover it was shown that pyruvate forms a covalent C-S bond at C221 and is thus converted to an enzyme bound hemithioketal. Apart from the core cysteine residue, there are a number of amino acid residues at the regulatory site of ScPDC located within the interaction distance of the covalently bound pyruvate (figure 2); three histidines (H92, H225, and H310), a glycine and an
ACS Paragon Plus Environment
4
Page 5 of 51
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
Biochemistry
adjacent alanine (positions 286 and 287), and a serine (position 311). Although side chain atoms of all these amino acid residues, including C221, are involved in the conformation transition from the catalytically inactive to the activated state of the enzyme as judged by their spatial shifts during this process25, the individual role of single residues, except for C221, remained largely elusive. To investigate these effects in more detail we generated a number of enzyme variants by site-directed mutagenesis. The histidines were changed to phenylalanines to suppress electrostatic interactions of the imidazole moieties with the carboxylate group of the regulatory substrate. Moreover, phenylalanine was chosen in order to maintain the sterical restrictions at the regulatory site. The functional groups of the serine and the cysteines (there is a neighboring at position 222) were eliminated, as well as the methyl moiety of the alanine. To determine the specific roles of individual amino acid residues in the process of allosteric substrate activation, the resulting variants have been kinetically analyzed. The obtained parameters have been interpreted in terms of physicochemical mechanisms.
THEORETICAL BASIS The kinetic model The minimal model of pyruvate decarboxylase action, comprising both the activation and the catalytic process can be written as 4-5, 30-32
ACS Paragon Plus Environment
5
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
Page 6 of 51
Scheme 1 In this scheme, species indexed with “i” refer to inactive enzyme forms, while species indexed with “a” refer to active enzyme forms. Within the framework of this model activation is represented by a two step mechanism leading from Ei through SEi to SEa 4-5, 30-32. The first step represents a rapid primary binding of the activating substrate molecule at the regulatory site. On the time scale of stopped-flow measurements this step cannot be resolved. Consequently, the step is assumed to be a rapid equilibrium. The rate limiting step of activation is the transition from the substrate bound but inactive species SEi to SEa. The corresponding rate constants are kiso and k-iso. The term “iso” refers to the structural isomerization process of the protein component necessarily involved in the activation process. The structural change itself is well documented by crystallographic and SAXS studies10-12, 22, 25, 33. As a consequence of the slow activation progress curves
of
substrate
[S ] = [S 0 ] − v SS ⋅ t +
consumption
v SS ⋅ 1 − e − kobs ⋅t k obs
(
are
described
by
)
the
following
equation
Equation 1
Here, vss represents the steady state velocity reached after the activation process has finished. [S0] is the initial substrate concentration. The rate constant kobs characterizes the time behavior of activation and depends on substrate concentration. Steady state kinetics The rate equation corresponding to the reaction scheme 1 under steady state conditions is: Vmax ⋅ [S ] = 2 A + B ⋅ [S ] + [S ] 2
vSS
Equation 2
with the observable steady state parameters
ACS Paragon Plus Environment
6
Page 7 of 51
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
Biochemistry
A = Ka ⋅ Kiso ⋅ Km
Equation 3
B = Km ⋅ (1 + Kiso )
Equation 4
Vmax =
k 2 ⋅ k3 ⋅ [E0 ] k 2 + k3
Equation 5
The appearance of quadratic substrate concentration terms in equation 2 is an expression of the positive cooperativity inherent in the mechanism and is responsible for the sigmoidity of the v/[S] plots. The catalytic cycle minimally involves the species SEa, SEaS and SEaP. The
equilibrium constants Ka and Kiso are defined as
Ka =
k− a ka
K iso =
Equation 6
k− iso kiso
Equation 7
The Km value is linked to the catalytic cycle and given by Km =
k3 (k−1 + k2 ) ⋅ k1 (k2 + k3 )
Equation 8
It follows from equation 2 that only the composite parameters A, B and Vmax can be determined from steady state measurements. Specifically, the Km value while being a factor of the observable steady state parameters A and B is not identical to the S0.5 value that is directly observable but also represents a composite value, given by equation 9. S0.5 =
B + 2
B2 +A 4
Equation 9
Transient kinetics
ACS Paragon Plus Environment
7
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
Page 8 of 51
The time-dependent behavior of the progress curves according to the mechanism in scheme 1 is represented by equation 1. The corresponding rate constant kobs is given by equation 10 (see also the appendix in the supplement section)
kobs =
kiso ⋅ [S ] k − iso ⋅ K m + K a + [S ] K m + [S ]
Equation 10
This relationship implies that kobs converges asymptotically to kiso at very high substrate concentrations, requiring that the conditions [S]>>Ka and [S]>>Km are simultaneously met. At very low substrate concentrations kobs converges to k-iso. This holds true under all experimental conditions and does not depend on the specific geometric character of the kobs/[S] plot. It can be shown analytically that kobs can run through extrema in dependence of [S] if certain algebraic conditions hold. The extremum is generally found at
[S ]ext =
K m ⋅ K a / (K iso ⋅ K m ) − K a
Equation 11
1 − K a / (K iso ⋅ K m )
It will correspond to a maximum of kobs if Km / Ka > Kiso > Ka / Km and to a minimum if Ka / Km > Kiso > Km / Ka . Otherwise [S]ext will be negative and therefore become irrelevant for real
data. Equation 1 strictly corresponds to the above minimal model assuming that the enzyme species labeled inactive before activation is indeed totally inactive. Relaxing this constraint one obtains instead
[S ] = [S 0 ] − vSS ⋅ t + (vSS − v0 ) ⋅ (1 − e −k k obs
obs ⋅t
)
Equation 12
Equation 12 formally expresses the consumption of substrate due to the enzymatic reaction. At zero time the rate of substrate conversion is determined by v0, while after infinitely long reaction
ACS Paragon Plus Environment
8
Page 9 of 51
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
Biochemistry
times the rate is given by vss. This corresponds to the transition from a low activity to a high activity state. In practice an observation window of 5-6 half lives is enough to reach the final steady state. In principle, equation 12 can also be applied in case of v0>vSS. For the wild type v0/vSS is very close to zero and can be safely neglected1. In case of variants, however, it is safe to include v0 during data evaluation30. Remarks on the internal relationships of kinetic parameters and potential pitfalls As our study mainly relies on the interpretation of kinetic data some cautionary remarks on internal relationships of the parameters as well as on their explanatory power might be in place. i) Steady state measurements: In case of ScPDC the activation mechanism leads to positive cooperativity in the steady state data. Thus observation of positive cooperativity is itself an appropriate criterion of the prevalence of activation also in the variants. Within the framework of the above model cooperativity is limited to apparent Hill coefficients between one and two. Using determined values of A, B and [S0.5] Hill coefficients nh can be estimated via nh =
2 ⋅ A + B ⋅ [S0.5 ] A + B ⋅ [S 0.5 ]
Equation 13
This corresponds to the following statements. Under the condition A
