Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.     

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.     

Synthesis and properties of 2-azido-NAD+. A study of interaction with glutamate dehydrogenase

A photoactive coenzyme analog of NAD+ has been synthesized by chemically coupling [32P]2-azido-AMP and NMN to produce [32P]nicotinamide 2-azidoadenosine dinucleotide (2-azido-NAD+). The utility of 2-azido-NAD+ as an effective active-site-directed photoprobe was demonstrated using bovine liver glutamate dehydrogenase as a model enzyme. In the absence of ultraviolet light, 2-azido-NAD+ is a substrate for this enzyme. Photoincorporation of probe was saturable with two different apparent dissociation constants of 10 microM and 40 microM. Protection of photoinsertion was seen with the natural substrate NAD+ with apparent dissociation constants of less than 5 microM and 25 microM. This observation may be explained on the basis of negative cooperative interaction between the subunits. The photoinsertion of 2-azido-NAD+ was increased by GTP and decreased by ADP in accordance with their known effects on NAD+ binding. When the enzyme was covalently modified by photolysis in the presence of saturating amounts of photoprobe, an approximately 40% inhibition of the enzyme activity was observed. These results demonstrate that the photoaffinity coenzyme analog has potential application as a probe to characterize NAD(+)-binding proteins and to identify the active sites of these proteins.     

Salicylate biosynthesis in Pseudomonas aeruginosa. Purification and characterization of PchB, a novel bifunctional enzyme displaying isochorismate pyruvate-lyase and chorismate mutase activities

Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of the pchB gene, was purified to homogeneity from Pseudomonas aeruginosa. In the reaction catalyzed by this enzyme, isochorismate --> salicylate + pyruvate, no cofactors appear to be required. At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent K(m) of 12.5 microm for isochorismate and a kcat of 106 min(-1), calculated per monomer. The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments. PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs). Chorismate was shown to be converted to prephenate by purified PchB in vitro, with an apparent K(m) of 150 microm and a kcat of 7.8 min(-1). An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB. Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P. aeruginosa pchB gene for growth in minimal medium. A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity. In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity.     

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.     

Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity

Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

1-Stearyl,2-stearoylaminodeoxy phosphatidylcholine, a potent reversible inhibitor of phospholipase A2

1-stearyl, 2-stearoylaminodeoxy phosphatidylcholine, a structurally modified phospholipid substrate analog exhibits potent and reversible inhibition of phospholipase A2 from cobra venom (N. naja naja). The apparent KI values determined in two different assay systems employing phosphatidylcholine-surfactant mixed micelles are in reasonable agreement (40 microM and 16 microM) and indicate that the inhibitor binds to the enzyme as much as two orders of magnitude more tightly than does dipalmitoyl phosphatidylcholine. With phosphatidylethanolamine as substrate, the kinetics are more complicated as the analog also exhibits activation, presumably at a second binding site on the enzyme.     

Purification and characterization of thymidylate synthetase from rat regenerating liver

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.     

Kinetic evidence of the existence of a regulatory phosphoenolpyruvate binding site in maize leaf phosphoenolpyruvate carboxylase

Phenylphosphate, a structural analog of phosphoenolpyruvate (PEP), was found to be an activator of phosphoenolpyruvate carboxylase (PEP carboxylase) purified from maize leaves. This finding suggested the presence in the enzyme of a regulatory site, to which PEP could bind. We carried out kinetic studies on this enzyme using controlled concentrations of free PEP and of Mg-PEP complex and developed a theoretical kinetic model of the reaction. In summary, the main conclusions drawn from our results, and taken as assumptions of the model, were the following: (i) The affinity of the active site for the complex Mg-PEP is much higher than that for free PEP and Mg2+ ions, and therefore it can be considered that the preferential substrate of the PEP-catalyzed reaction is Mg-PEP. (ii) The enzyme has a regulatory site specific for free PEP, to which Mg2+ ions can not bind. (iii) The binding of free PEP, or an analog molecule, to this regulatory site yields a modified enzyme that has much lower apparent Km values and apparent Vmax values than the unmodified enzyme. So, free PEP behaves as an excellent activator of the reaction at subsaturating substrate concentrations, and as an inhibitor at saturating substrate concentrations. These findings may have important physiological implications on the regulation of the PEP carboxylase in vivo activity and, consequently, of the C4 pathway, since increased reaction rates would be obtained when the concentration of PEP rises, even at limiting Mg2+ concentrations.     

Biosynthesis of isochorismate in Klebsiella pneumoniae: origin of O-2

The shikimate metabolites are key precursors to a large number of natural products, including aromatic amino acids. Chorismic acid is an important branch point in the biosynthetic pathway to aromatic amino acids. Chorismic acid is also unique among natural products since it is the only compound known to undergo an enzymatic Claisen rearrangement. A metabolite of chorismic acid, isochorismic acid, first observed in Aerobacter aerogenes differs in its chemical structure by the location of the hydroxyl group and the double bonds. Isochorismic acid is a precursor to a growing number of shikimate-derived metabolites. Isochorismic acid has also been postulated to be an intermediate of m-carboxyaromatic amino acids, implying another enzymatic Claisen rearrangement. In this publication, we have isolated isochorismate synthase and found that on lyophilization the enzyme is stable for at least 6 months at -20 degrees C. Incubation of chorismate with this preparation in water enriched with 18O led to incorporation of one atom of 18O as proven from the fast atom bombardment mass spectra of the HPLC purified derived isochorismate.     

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.     

Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader.

The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1. The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.     

Implications of the role of reactive cystein in arginine kinase: reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol

The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.     

The mode of binding of potential transition-state analogs to acetylcholinesterase.

Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Structure and mechanism of MbtI, the salicylate synthase from Mycobacterium tuberculosis

MbtI (rv2386c) from Mycobacterium tuberculosis catalyzes the initial transformation in mycobactin biosynthesis by converting chorismate to salicylate. We report here the structure of MbtI at 2.5 A resolution and demonstrate that isochorismate is a kinetically competent intermediate in the synthesis of salicylate from chorismate. At pH values below 7.5 isochorismate is the dominant product while above this pH value the enzyme converts chorismate to salicylate without the accumulation of isochorismate in solution. The salicylate and isochorismate synthase activities of MbtI are Mg2+-dependent, and in the absence of Mg2+ MbtI has a promiscuous chorismate mutase activity similar to that of the isochorismate pyruvate lyase, PchB, from Pseudomonas aeruginosa. MbtI is part of a larger family of chorismate-binding enzymes descended from a common ancestor (the MST family), that includes the isochorismate synthases and anthranilate synthases. The lack of active site residues unique to pyruvate eliminating members of this family, combined with the observed chorismate mutase activity, suggests that MbtI may exploit a sigmatropic pyruvate elimination mechanism similar to that proposed for PchB. Using a combination of structural, kinetic, and sequence based studies we propose a mechanism for MbtI applicable to all members of the MST enzyme family.     

The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product. Evidence that EntA does not contribute to isochorismate synthase activity

The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z. Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate. Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction. Addition of EntA had no effect on isochorismate synthase activity. As a result, the mutation (previously designated entC401) in strain AN191 was characterized by nucleotide sequence analysis. The lesion is a single base substitution in the entA gene, resulting in a glutamic acid-for-glycine substitution at the penultimate amino acid (residue 247) of the EntA enzyme. The mutant protein was partially purified and shown to be devoid of 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase activity, whereas the entC gene product from strain AN191 exhibited normal isochorismate synthase function. These results conflict with the earlier characterization of the entC401 mutation in a different genetic background. The data presented herein establish that the EntA protein does not contribute to isochorismate synthase activity and that the mutant strain that led to this suggestion harbors a defective allele of entA rather than entC.     

Purification and characterization of a kallikrein from human submaxillary glands

A tissue kallikrein was purified over 1500-fold from the postmicrosomal supernatant of human submaxillary glands. The purified enzyme gave a single band, corresponding to an apparent molecular weight of 42,000 on SDS-polyacrylamide gel electrophoresis. This enzyme cross-reacted with the anti-human urinary kallikrein antiserum. The purified enzyme was characterized in comparison with the purest human urinary kallikrein preparation. Both enzymes hydrolyzed the synthetic substrate, Ac-Phe-Arg-OMe, most effectively. Aprotinin, TLCK, and PMSF suppressed the enzyme activities, while SBTI, LBTI, and alpha 1-antitrypsin had no effect at all. The purified enzyme generated kinin from the natural substrate, kininogen. It was concluded therefore that the purified enzyme is a typical tissue kallikrein.