University of Richmond Department of Chemistry: Biochemistry & Molecular Biology Program

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ASBMB 2002

Probing the Active Site of 3-phosphoglycerate Dehydrogenase: The Role of Acid Base Catalysis and the Local Charge Environment of the Transition State in V Type Regulation by Serine

Donnie Berkholz# and Ellis Bell*Gustavus Adolphus College# and University of Richmond*

Abstract3-Phosphoglycerate Dehydrogenase catalyzes a hydride transfer reaction that utilizes NAD(H) depending upon the direction of hydride transfer and interconverts 3-phosphoglycerate and Phosphohydroxypyruvate. The enzyme is part of the serine biosynthetic pathway and is subjected to V-type allosteric regulation by serine. The crystal structure of the serine inhibited conformation of the enzyme reveals the presence of an active site histidine, H292 and three negatively charged residues, D264, E269 and E213, surrounding the nicotinamide ring of the cofactor. To investigate both the role of the histidine in catalysis and the potential roles of the negative charged residues in either orienting or changing the basicity of the imidazole ring, each of the above residues has been subjected to site directed mutagenesis. The four mutant proteins, H292Q, E264Q, E213Q and D269N have been overexpressed, purified and characterized and compared with native protein for rates of hydride transfer, Km, overall conformation, quaternary structure and stability. H292Q reduced kcat by a factor of 102 but had essentially unaltered conformation, stability and cofactor binding capacity. Both E269Q and D264N resulted in significant decreases in kcat, by factors of up to 2 x 102. While E269Q showed unaltered cofactor binding, D264N was essentially free of cofactor as isolated, suggesting a role for D264 in both catalysis and cofactor binding. E213Q showed native catalytic activity but had significantly reduced stability. All four mutants and the native protein, when saturated with NADH, showed the characteristic resonance energy transfer from reduced cofactor to a nearby tryptophan residue, indicating that the active site geometry was essentially unchanged in all the mutants. The data demonstrate that the catalytic effectiveness of the active site histidine and the overall reaction is modulated by both E269 and D264. Examination of the crystal structure suggests that D264 must rotate into the active site vicinity in the uninhibited native enzyme to account for these findings. The magnitude of the effects of this rotation into [uninhibited] or out of [serine inhibited] the active site is sufficient to account for the magnitude of serine inhibition. This is confirmed by the fact that while the other mutants and native PGDH are potently inhibited by serine, the D264N mutant shows very significantly decreased inhibition by serine even at 1mM. This work was supported by NSF Grant etc

3-Phosphoglycerate Dehydrogenase

Specific Aims and Approach

Experimental Approaches:Construction and Expression of Site Directed Mutants

Site directed mutants were constructed using oligonucleotide directed mutagenesis via the Stratagene QuikChange™ mutagenesis protocols. The mutated vector was transformed into competent DH5 [PGDH-] cells and plated onto LB agar plates containing ampicillin. Starter cultures, using individual colonies from the plates, were grown at 37ºC to turbidity and transferred to 1l flasks of LB media with 100/mL ampicillin. At an OD600nm of ~0.6, the cultures were then induced with 1mM IPTG and grown for a further 6 hours. Cell pellets were harvested by centrifugation and resuspended in 50mM KH2PO4, 2mM DTT, 1mM EDTA and a cocktail of protease inhibitors to approximately 5mL/gm of cells. Cells were lysed by sonication.

Subsequent purification of the mutant protein involved polyethyleneimine precipitation of DNA, and fractional precipitation of protein by AmSO4 cuts [10, 30 and 70%] with final chromatography of the resuspended 70% AmSO4 pellet on Sephacel S200. The protein from the gel filtration chromatography was pure as judged by SDS-PAGE.

HINT Analysis of Cofactor Binding

Kinetic Properties of the MutantsMore detailed kinetic studies included determination of Km for both substrates from Lineweaver-Burke plots with either NADH or ketoglutarate as the varied substrate for native and each mutant enzyme. No significant differences were found for Km for either substrate between the native and any mutant indicating that Vmax was the only kinetic property significantly changed by the mutations. Activation energies for the catalyzed reaction were determined from the temperature dependence of Vmax. Values of 1.02Kcals/Mole, 2.9Kcals/Mole, 2.5Kcals/Mole and 4.1Kcals/Mole were obtained for Native, E213Q, H292Q, E269Q and D264N forms respectively.

Resonance Energy Transfer to Bound Cofactor

Activation Energy Analysis Serine Regulation of the Native and Mutant Forms

Conclusions:

Acid-Base Catalysis and H292The fact that the H292Q mutant still shows significant catalytic activity suggests that while Acid Base catalysis plays a significant role in the reaction catalyzed by 3-phosphoglycerate dehydrogenase, it is not the only factor contributing to chemical catalysis in the active site.

The Role of E269 Although based upon the crystal structure of the serine inhibited enzyme, it was postulated that E269 played a role in changing the basicity of H292 it would appear that this effect is minimal compared to the influences on the catalytic process. Whether E269 does in fact change the basicity of H292 or contributes in other ways to catalysis is unclear.

The Role of D264 D264 clearly plays a critical role in cofactor binding but also a major role in catalysis in 3-phosphoglycerate dehydrogenase. Mutation to asparagine causes not only the greatest change in Vmax of the mutations studied here but also causes a significant change in the affinity of the enzyme for cofactor. This change is of such magnitude that the mutant protein is bound without cofactor bound, unlike either the native protein or the other mutants. Whether D264 plays a role in changing the basicity of H292 remains to be determined. By rotation into the active site, instead of its location in the serine inhibited enzyme, it could influence the basicity of H292. Alternatively, it could be the major contributor to the overall negative charge environment around the catalytic center of the enzyme which could be playing an indirect role in catalysis by modulating the dynamic structure of the protein effecting the efficiency of hydride tunneling.

D264 and Serine Regulation

Irrespective of the role that D264 plays in catalysis, it is clear that it plays a major role in the V type allosteric regulation of the enzyme by serine. The major portion of the Vmax change ellicited by serine is attributable to movement of D264 into (active conformation) and out of (serine inhibited conformation) the active site cavity. How serine binding to the regulatory domain, 33Å from the active site, triggers this conformational changes remains to be determined.

Introduction and Background:

3-phosphoglycerate dehydrogenase, EC 1.1.1.95 is involved in the biosynthesis of serine and as a result plays an important role in both phospholipid and tryptophan metabolism.

The enzyme is a tetramer, which in prokaryotic cells is allosterically regulated by the end product of the biosynthetic pathway, serine. The three dimensional structure of the E Coli enzyme reveals a multidomain subunit with separate cofactor and substrate domains and a serine binding regulatory domain1. In eukaryotic systems the activity of the enzyme is regulated by transcriptional control2. Analysis of sequence alignments of a variety of prokaryotic and eukaryotic sequences suggests that the eukaryotic enzymes have conserved sequence in the region of all three domains of the E Coli enzyme but have an insert between the substrate and regulatory domains. While the eukaryotic enzymes are not allosterically regulated by serine, serine does bind and affect their thermal stability.

Catalytic MechanismThe presumed catalytic mechanism of the enzyme involves the transfer of a hydride anion from the substrate to the nicotinamide ring of the cofactor and the base catalyzed abstraction of a proton from the -OH of the substrate to the nitrogen of the imidazole ring of Histidine 292 on the protein. It has been presumed that the basicity of this histidine is increased by the proximity, in the active site, of Glutamate 269. Visual analysis of the active site region of the enzyme shows that there are in fact three carboxyl side chains within 5-8Å of the location of the chemical transformation, E269, E213 and D264, creating an unusually negatively charged environment for catalysis relative to other dehydrogenases catalyzing similar reactions [eg MDH].

The specific aims for this project were

1] To examine the impact that the charge environment around the active site has on the catalytic ability of the enzyme.

2] To investigate whether modulation of this negatively charged environment is involved in the V type allosteric regulation of the enzyme by serine.

To achieve these goals, the three carboxyl side chains within 5-8Å of the active site, E269, E213 and D264, have been mutated to the appropriate amide and the presumed catalytic base, Histidine 292, has been mutated to glutamine. The effects of the mutations on both catalysis and cofactor binding/conformation were assessed.

To determine whether the mutants were still sensitive to serine inhibition, preliminary screenings were done with native and mutant enzymes using no serine, 100M and 1000M serine, levels which with the native enzyme give effectively complete inhibition. As shown above, E213Q showed normal serine inhibition, H292Q and E269Q showed small deviations from the native pattern of serine inhibition but D264N showed a major difference. As shown below with a more complete range of serine concentrations, the D264N mutant showed at saturating serine concentrations 30% residual activity compared to 1% or less for the native enzyme. The D264N mutation clearly has a major impact on the ability of serine to inhibit the enzyme. This suggests that the carboxyl group of D264 is intimately involved in the mechanism of serine inhibition.

Native 3-phosphoglycerate Dehydrogenase shows a resonance energy transfer of fluorescence excitation of the single tryptophan per subunit to the NADH bound to an adjacent subunit resulting in decreased tryptophan fluorescence at 335nm and sensitized NADH fluorescence at 450nm. When native enzyme, or the mutants, were titrated with aliquots of NADH both the quenching of the tryptophan fluorescence and the sensitization of the NADH fluorescence were virtually identical, as shown above for the D264N mutant. Similar effects were observed for each of the mutants. Since resonance energy transfer effects are extremely sensitive to both the orientation and distance of separation of the donor (tryptophan) and acceptor (NADH), these observations suggest that the overall active site geometry of the mutant enzymes is similar to that of the native enzyme.

The Mechanism of Allosteric Regulation of

3-phosphoglycerate Dehydrogenase.

3-p-GDH shows a V-type allosteric regulation, with serine binding to the regulatory domain and affecting Vmax rather than Km for either substrate3. Each Serine binding involves residues from both polypeptide chains at the interface of the regulatory domains of the molecule: a total of 4 serine molecules bind. Serine binding exhibits positive cooperativity for the first two molecules of serine to bind and negative cooperativity for subsequent serine binding4. The molecular mechanism of serine inhibition is currently unknown, but clearly requires a detailed understanding of the active site geometry and the role that individual residues in the active site play in the overall catalytic mechanism of the enzyme.

The Active Site Cavity

Shown above is the active site cavity of the enzyme with the presumed catalytic base, H292, and three adjacent carboxyl groups, E269, E213 and D264 labeled. NAD is also shown in space fill representation. From this perspective the substrate, 3-phosphoglycerate binds above and to the left of the NADH/H292 location.

Characterization of Native and Mutant ProteinsAll mutant proteins were characterized for both physical and kinetic properties for comparison with native protein. UV spectra revealed that, like the native protein, the H292Q, E213Q and E269Q mutants had tightly bound cofactor resulting in a 280:260 ratio of less than one. The D264N mutant showed a 280:260 ratio of 1.6 suggesting a lack of tightly bound cofactor. This was confirmed by chemical denaturation using tri-chloroacetic acid and spectral analysis of released nucleotide.

Specific Activity measurements were performed using saturating concentrations of both NADH and the alternative substrate ketoglutarate at concentrations of either 0.1mM and 1mM or 0.2mM and 2mM, respectively. Similar values for the specific activity were obtained with either conditions indicating that the assay conditions were indeed saturating.

Protein concentrations were determined using a calibrated Bradford Dye Binding assay since the variable amounts of bound nucleotide indicated by the 280:260 ratios would interfere with the accurate determination of the protein concentration by A280nm measurements. Since the A280:260 ratio measurements suggested that cofactor binding was significantly affected by

the D264N mutation, cofactor binding was subjected to HINT analysis5, performed in conjunction with Dr. James Burnett, VCU to examine specific contributions to cofactor binding. This analysis, using the crystal coordinates of the serine inhibited enzyme indicated that D264 made a hydrogen bond interaction with the NH2 proton of the cofactor. In this location (see “A” below)), D264 is located too far from the catalytic Histidine to exert influence over its basicity. If, however, D264 is rotated about its CC bond which is sterically allowed in the crystal structure to a position where the other oxygen (see “B”) interacts with the cofactor NH2, the charge of the group is brought into closer proximity to the Histidine ring and could now exert a charge effect on H292.

A B

This analysis, in conjunction with the experimental results obtained here for the D264N mutation suggest that in the native protein D264 is rotated into the active site cavity where it can still hydrogen bond to the NH2 of the cofactor but also influence the local charge environment of the catalytic base H292: mutation to Asparagine would negate both effects as experimentally observed.

References1. Schulller, D. Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2:69-76.2. Achouri, Y., Rider, M. H., Schaftingen, E. V., and Robbi, M. (1997) Biochem. J. 323:365-370.3. Sugimoto, E., and Pizer, L. (1968) J. Biol. Chem. 243:2081-2089.4. Grant, G. A., Schuller, D. J., Banaszak, L. J. (1996) Prot. Sci. 5:34-41.5. Kellogg, G. E., Semus, S. F. Abraham, D. J. (1991) J. Comput. Aided Mol. Des. 5:545-552.