Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

"Catch 222," the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69

R67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in Km values for both ligands and a 2-fold rise in the kcat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344-11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the Kd and Km values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type Km value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate.     

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.     

Multiple roles of arginine 181 in binding and catalysis in the NAD-malic enzyme from Ascaris suum

The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.     

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.     

The influence of conserved aromatic residues in 3-hydroxy-3-methylglutaryl-CoA synthase

In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins. Each mutant protein was expressed at levels comparable with wild-type enzyme and was isolated in highly purified form. Initial kinetic characterization indicated that F204L exhibits a substantial (>300-fold) decrease in catalytic rate (kcat). Upon modification with the mechanism-based inhibitor, 3-chloropropionyl-CoA, or in formation of a stable binary complex with acetoacetyl-CoA, F204L exhibits binding stoichiometries comparable with wild-type enzyme, suggesting substantial retention of active site integrity. Y130L and Y376L exhibit inflated values (80- and 40-fold, respectively) for the Km for acetyl-CoA in the acetyl-CoA hydrolysis partial reaction; these mutants also exhibit an order of magnitude decrease in kcat. Formation of the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and Y376L proceeds slowly in comparison with wild-type enzyme. However, solvent exchange into the thioester carbonyl oxygen of these acetyl-S-enzyme intermediates is not slow in comparison with previous observations for D159A and D203A mutants, which also exhibit slow acetyl-S-enzyme formation. The magnitude of the differential isotope shift upon exchange of H218O into [13C]acetyl-S-enzyme suggests a polarization of the thioester carbonyl and a reduction in bond order. Such an effect may substantially contribute to the upfield 13C NMR shift observed for [13C]acetyl-S-enzyme. The influence on acetyl-S-enzyme formation, as well as observed kcat (F204L) and Km (Y130L; Y376L) effects, implicate these invariant residues as part of the catalytic site. Substitution of phenylalanine (Y130F, Y376F) instead of leucine at residues 130 and 376 diminishes the effects on catalytic rate and substrate affinity observed for Y130L and Y376L, underscoring the influence of aromatic side chains near the active site.     

Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib.

The activation of human platelets by alpha-thrombin is mediated in part by cleavage of the protease-activated receptor (PAR) 1 and 4 and by the glycoprotein (Ib)alpha, (Gp(Ib)alpha), which binds with high affinity to alpha-thrombin. Recent studies have shown that the thrombin domain referred to as heparin binding site (HBS) is involved in the interaction with the platelet Gp(Ib)alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to Gp(Ib)alpha, we have performed alanine scanning mutagenesis of the basic HBS R93, R97, R101, R233, K236, K240, R233/K236/Q239, as well as of the neutral Q239 residues, located in different regions of the domain. For comparison, mutation at R67 within the fibrinogen recognition site (FRS) of thrombin was performed as well. Solid-phase binding experiments showed that the Kd of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutant R233A/K236A/Q239A, while the Q239A and R67A forms did not show any significant affinity change. The platelet activating capacity of these mutants was evaluated as well. Using gel-filtered platelets, the EC50 value of thrombin-induced aggregation was from 5- to 13-fold higher in the HBS mutants than in the WT form, and was linearly and positively correlated with the corresponding Kd values pertaining to thrombin binding to GpIb. Measurements of PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R233A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the apparent kcat/Km value. These results are a consequence of a defective binding to GpIb, which is known to optimize the interaction with PAR-1 in situ. A confirm of this hypothesis came from the demonstration that the kcat/Km value pertaining to the hydrolysis by the HBS-mutated thrombins of the synthetic PAR-1 38-60 peptide in solution was similar to that one obtained with the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding to the platelet Gp(Ib)alpha and in the cell activation.     

Analysis of the leukemia inhibitory factor receptor functional domains by chimeric receptors and cytokines

In contrast to other hematopoietic cytokine receptors, the leukemia inhibitory factor receptor (LIFR) possesses two cytokine binding modules (CBMs). Previous studies suggested that the NH(2)-terminal CBM and the Ig-like domain of the LIFR are most important for LIF binding and activity. Using the recently engineered designer cytokine IC7, which induces an active heterodimer of the LIFR and gp130 after binding to the IL-6R, and several receptor chimeras of the LIFR and the interleukin-6 receptor (IL-6R) carrying the CBM of the IL-6R in place of the COOH-terminal LIFR CBM, we could assign individual receptor subdomains to individual binding sites of the ligand. The NH(2)-terminal CBM and the Ig-like domain of the LIFR bind to ligand site III, whereas the COOH-terminal CBM contacts site I. Furthermore, we show that LIFR mutants carrying the IL-6R CBM instead of the COOH-terminal CBM can replace the IL-6R by acting as an alpha-receptor for IL-6. However, in situations where a signaling competent receptor is bound at IL-6 site I, ligand binding to site III is an absolute requirement for participation of the receptor in a signaling heterodimer with gp130; i.e., a functional receptor complex of IL-6 type cytokines cannot be assembled solely via site I and II as in the growth hormone receptor complex.     

Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding.

The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.     

Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles

Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.     

Substrate and metal complexes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae provide new insights into the catalytic mechanism

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent enzymes that catalyse the first committed step in the biosynthesis of aromatic amino acids in microorganisms and plants, the condensation of 2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP synthases are possible targets for fungicides and represent a model system for feedback regulation in metabolic pathways. To gain further insight into the role of the metal ion and the catalytic mechanism in general, the crystal structures of several complexes between the tyrosine-regulated form of DAHP synthase from Saccharomyces cerevisiae and different metal ions and ligands have been determined. The crystal structures provide evidence that the simultaneous presence of a metal ion and PEP result in an ordering of the protein into a conformation that is prepared for binding the second substrate E4P. The site and binding mode of E4P was derived from the 1.5A resolution crystal structure of DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde 3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl group of E4P replaces a water molecule coordinated to the metal ion, strongly favouring a reaction mechanism where the initial step is a nucleophilic attack of the double bond of PEP on the metal-activated carbonyl group of E4P. Mutagenesis experiments substituting specific amino acids coordinating PEP, the divalent metal ion or the second substrate E4P, result in stable but inactive Aro4p-derivatives and show the importance of these residues for the catalytic mechanism.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.     

Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase

The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.     

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. On the basis of this precedent, conserved basic residues of human PMK have been mutated, and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60- and 30-fold, respectively) as well as decreases [50-fold (forward) and 85-fold (reverse)] in Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax [14-fold (forward) and 10-fold (reverse)] but has inflated Km values for ATP and ADP (48- and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.     

The intrinsic ATPase activity of protein kinase C is catalyzed at the active site of the enzyme.

We recently reported that autophosphorylated protein kinase C (PKC) has an intrinsic Ca(2+)- and phospholipid-dependent ATPase activity and that the ATPase and histone kinase activities of PKC have similar metal-ion cofactor requirements and Km,app(ATP) values. We hypothesized that the intrinsic ATPase activity of PKC may represent the bond-breaking step of its protein kinase activity. The rate of the ATPase reaction is several times slower than the histone kinase reaction rate. At subsaturating concentrations, various peptide and protein substrates stimulate the ATPase reaction by as much as 1.5-fold. In contrast, non-phosphorylatable substrate analogs are not stimulatory. These observations support a mechanism of PKC catalysis in which the productive binding of phosphoacceptor substrates enhances the rate of phosphodonor substrate (ATP) hydrolysis at the active site of PKC. However, this mechanism contains an assumption that the ATPase activity of PKC is catalyzed at the active site. In fact, sequence analysis indicates that PKC contains a potential second nucleotide binding site outside of its active site. In this report, we provide a detailed analysis of the relationship between the active site of PKC and the intrinsic ATPase activity of the enzyme. We show that the regulatory and catalytic properties of the ATPase reactions of three PKC isozymes are similar, despite critical differences among the isozymes in their consensus sequences for the potential non-active-site nucleotide binding site in their catalytic domains. We also show that the ATPase and histone kinase reactions of each isozyme have similar Km,app(ATP) values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a novel cofactor containing N-(7-mercaptoheptanoyl)-O-3-phosphothreonine

The cofactor required in the methylcoenzyme M methylreductase reaction was shown to be a large molecule with an Mr of 1149.21 in the free acid form. The cofactor, named MRF for methyl reducing factor, was identified from analyses by fast atom bombardment mass spectrometry and 1H, 13C, and 31P NMR spectroscopy as uridine 5'-[N-(7-mercaptoheptanoyl)-O-3-phosphothreonine-P-yl(2-acetamido- 2-deoxy- beta-mannopyranuronosyl)(acid anhydride)]-(1----4)-O-2-acetamido-2-deoxy- alpha-glucopyranosyl diphosphate. MRF contains N-(7-mercaptoheptanoyl)threonine O-3-phosphate (HS-HTP) [No11, K. M., Rinehart, K. L., Tanner, R. S., & Wolfe, R. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242] and is linked to C-6 of 2-acetamido-2-deoxymannopyranuronic acid of the UDP-disaccharide through a carboxylic-phosphoric anhydride linkage. It is postulated that this bond is responsible for the instability of the molecule and its hydrolysis during isolation. Analyses of Eadie and Hofstee plots of the methylcoenzyme M methylreductase reaction indicate that MRF has a 6-fold lower Km(app) than HS-HTP and a 50% greater Vmax. This suggests that the UDP-disaccharide moiety may be of importance in the binding of MRF to the enzyme active site.     

The influence of basic residues on the substrate specificity of protein kinase C

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.