Presence of 18-A long hydrogen bond track in the active site of Escherichia coli DNA polymerase I (Klenow fragment). Its requirement in the stabilization of enzyme-template-primer complex

The analysis of the active site region in the crystal structures of template-primer-bound KlenTaq (Klenow fragment equivalent of Thermus aquaticus polymerase I) shows the presence of an approximately 18-A long H-bonding track contributed by the Klenow fragment equivalent of Asn(845), Gln(849), Arg(668), His(881), and Gln(677). Its location is nearly diagonal to the helical axis of the template-primer. Four base pairs in the double stranded region proximal to 3' OH end of the primer terminus appear to interact with individual amino acid components of the track through either the bases or sugar moieties. To understand the functional significance of this H-bonding network in the catalytic function of Klenow fragment (KF), we generated N845A, N845Q, Q849A, Q849N, R668A, H881A, H881V, Q677A, and Q677N mutant species by site-directed mutagenesis. All of the mutant enzymes showed low catalytic activity. The kinetic analysis of mutant enzymes indicated that K(m)(.dNTP) was not significantly altered, but K(D)(.DNA) was significantly increased. Thus the mutant enzymes of the H-bonding track residues had decreased affinity for template-primer, although the extent of decrease was variable. Most interestingly, even the reduced binding of TP by the mutant enzymes occurs in the nonproductive mode. These results demonstrate that an H-bonding track is necessary for the binding of template-primer in the catalytically competent orientation in the pol I family of enzymes. The examination of the interactive environment of individual residues of this track further clarifies the mode of cooperation in various functional domains of pol I.     

Role of Asp187 and Gln190 in the Na+/proline symporter (PutP) of Escherichia coli

Asp187 and Gln190 were predicted as conserved and closely located at the Na(+) binding site in a topology and homology model structure of Na(+)/proline symporter (PutP) of Escherichia coli. The replacement of Asp187 with Ala or Leu did not affect proline transport activity; whereas, change to Gln abolished the active transport. The binding affinity for Na(+) or proline of these mutants was similar to that of wild-type (WT) PutP. This result indicates Asp187 to be responsible for active transport of proline without affecting the binding. Replacement of Gln190 with Ala, Asn, Asp, Leu and Glu had no effect on transport or binding, suggesting that it may not have a role in the transport. However, in the negative D187Q mutant, a second mutation, of Gln190 to Glu or Leu, restored 46 or 7% of the transport activity of WT, respectively, while mutation to Ala, Asn or Asp had no effect. Thus, side chain at position 190 has a crucial role in suppressing the functional defect of the D187Q mutant. We conclude that Asp187 is responsible for transport activity instead of coupling-ion binding by constituting the translocation pathway of the ion and Gln190 provides a suppressing mutation site to regain PutP functional activity.     

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.     

Contribution of polar residues of the J-helix in the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I (Klenow fragment): Q677 regulates the removal of terminal mismatch

Previous structural and biochemical data indicate a participation of the J-helix of Escherichia coli pol I in primer positioning at the polymerase and exonuclease sites. The J-helix contains three polar residues: N675, Q677, and N678. Preliminary characterization of alanine substitutions of these residues showed that only Q677A DNA polymerase has substantially decreased polymerase and increased exonuclease activity. The Q677A enzyme had approximately 2- and approximately 5-fold greater exonuclease activity than the wild type (WT) with mismatched and matched template-primers (TPs), respectively. N675A and N678A DNA polymerases did not differ significantly from the WT in these activities, despite the fact that both residues are seen to interact with the TP in various pol I-DNA complexes. Pre-steady-state kinetic measurements for the exonuclease activity of WT and mutant enzymes indicated nearly identical DNA binding affinity for ssDNA and mismatched TPs. However, with a matched TP, Q677A DNA polymerase exhibited increased exonuclease site affinity. The most important characteristic of Q677A DNA polymerase was its ability to continue cleavage into the matched region of the TP after mismatch excision, in contrast to the WT and other mutant enzymes. The increase in the exonuclease activity of Q677A DNA polymerase was further determined not to be solely due to the weakened binding at the polymerase site, by comparison with another polymerase-defective mutant enzyme, namely, R668A DNA polymerase. These enzymes have significantly decreased DNA binding affinity at the polymerase site, yet the exonuclease activity parameters of R668A DNA polymerase remain similar to those of the WT. These results strongly suggest that participation of Q677 is required for positioning the primer terminus (a) in the polymerase site for continued nucleotide addition and (b) in the 3'-exonuclease site for the controlled removal of mismatched nucleotides.     

Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases

Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

The role of a conserved histidine residue, His324, in Trigonopsis variabilisd-amino acid oxidase

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.     

Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.     

Active site residues in Mycobacterium tuberculosis pantothenate synthetase required in the formation and stabilization of the adenylate intermediate

Pantothenate synthetase (EC 6.3.2.1) catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine in bacteria, yeast, and plants. The three-dimensional structural determination of pantothenate synthetase from Mycobacterium tuberculosis has indicated specific roles for His44, His47, Asn69, Gln72, Lys160, and Gln164 residues in the binding of substrates and the pantoyl adenylate intermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Ala mutants and determined their catalytic properties. The substitution of alanine for H44, H47, N69, Q72, and K160 residues in M. tuberculosis pantothenate synthetase caused a greater than 1000-fold reduction in enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolated adenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of one of these six amino acids with alanine, suggesting that these residues are essential for the formation of the pantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and beta-alanine in the second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutants and was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedly enhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of the K160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstrated that the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme for ATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residues are essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenate synthetase reaction.     

Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.     

Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand

In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.     

Role of Q52 in catalysis of decarboxylation and transamination in dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a pyridoxal phosphate dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. DGD uses stereoelectronic effects to control its unusual reaction specificity. X-ray crystallographic structures of DGD suggest that Q52 is important in maintaining the substrate carboxylate in a stereoelectronically activated position. Here, the X-ray structures of the Q52A mutant and the wild type (WT) DGD-PMP enzymes are presented, as is the analysis of steady-state and half-reaction kinetics of three Q52 mutants (Q52A, Q52I, and Q52E). As expected if stereoelectronic effects are important to catalysis, the steady-state rate of decarboxylation for all three mutants has decreased significantly compared to that of WT. Q52A exhibits an approximately 85-fold decrease in k(cat) relative to that of WT. The rate of the decarboxylation half-reaction decreases approximately 10(5)-fold in Q52I and approximately 10(4)-fold in Q52E compared to that of WT. Transamination half-reaction kinetics show that Q52A and Q52I have greatly reduced rates compared to that of WT and are seriously impaired in pyridoxamine phosphate (PMP) binding, with K(PMP) at least 50-100-fold greater than that of WT. The larger effect on the rate of l-alanine transamination than of pyruvate transamination in these mutants suggests that the rate decrease is the result of selective destabilization of the PMP form of the enzyme in these mutants. Q52E exhibits near-WT rates for transamination of both pyruvate and l-alanine. Substrate binding has been greatly weakened in Q52E with apparent dissociation constants at least 100-fold greater than that of WT. The rate of decarboxylation in Q52E allows the energetic contribution of stereoelectronic effects, DeltaG(stereoelectronic), to be estimated to be -7.3 kcal/mol for DGD.     

The ubiquinone-binding site of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase is a source of superoxide

The mitochondrial succinate dehydrogenase (SDH) is a tetrameric iron-sulfur flavoprotein of the Krebs cycle and of the respiratory chain. A number of mutations in human SDH genes are responsible for the development of paragangliomas, cancers of the head and neck region. The mev-1 mutation in the Caenorhabditis elegans gene encoding the homolog of the SDHC subunit results in premature aging and hypersensitivity to oxidative stress. It also increases the production of superoxide radicals by the enzyme. In this work, we used the yeast succinate dehydrogenase to investigate the molecular and catalytic effects of paraganglioma- and mev-1-like mutations. We mutated Pro-190 of the yeast Sdh2p subunit to Gln (P190Q) and recreated the C. elegans mev-1 mutation by converting Ser-94 in the Sdh3p subunit into a glutamate residue (S94E). The P190Q and S94E mutants have reduced succinate-ubiquinone oxidoreductase activities and are hypersensitive to oxygen and paraquat. Although the mutant enzymes have lower turnover numbers for ubiquinol reduction, larger fractions of the remaining activities are diverted toward superoxide production. The P190Q and S94E mutations are located near the proximal ubiquinone-binding site, suggesting that the superoxide radicals may originate from a ubisemiquinone intermediate formed at this site during the catalytic cycle. We suggest that certain mutations in SDH can make it a significant source of superoxide production in mitochondria, which may contribute directly to disease progression. Our data also challenge the dogma that superoxide production by SDH is a flavin-mediated event rather than a quinone-mediated one.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.     

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.     

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.     

Distinct conformation-mediated functions of an active site loop in the catalytic reactions of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase

The three-dimensional structures of NAD-dependent D-lactate dehydrogenase (D-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn97 and Glu141, which are also located at equivalent positions in D-LDH and FDH, respectively. X-ray crystallography at 2.4-A resolution revealed that replacement of Asn97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn97-->Asp mutant D-LDH exhibited virtually the same k(cat), but about 70-fold higher K(M) value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the K(M) value, but 110 and 590-fold decreases in the k(cat) values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141-->Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu141. These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning.