The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The catalytic mechanism of EPSP synthase revisited.

Recent analysis of EPSP synthase by solid-state NMR has led to the postulation of a new enzyme reaction pathway and raised once again the question of an intermediate species covalently bound to the enzyme [Studelska, D., McDowell, L., Espe, M., Klug, C., and Schaefer, J. (1997) Biochemistry 36, 15555-15560]. Therefore, we have reexamined the mechanism of the reaction catalyzed by EPSP synthase and analyzed the reaction products formed under the conditions used in preparing samples for solid-state NMR. Single-turnover experiments were carried out using both [1-14C]- and [32P]PEP showing the formation and decay of the previously proposed tetrahedral intermediate species on a time scale comparable with the disappearance of substrate and formation of product, thus unequivocally establishing the kinetic competence. The possible presence of a covalently bound enzyme intermediate species was also investigated, using SDS-PAGE and Centricon concentration analysis of the quenched reaction samples. No covalently bound enzyme intermediates were observed during the reaction. An enzyme assay was also performed repeating the conditions used in sample preparation for the solid-state NMR studies. We show that under these conditions, total turnover of substrates to products was observed within 45 s at -30 degrees C prior to freezing and lyophilization. Following lyophilization, the samples were stored at -20 degrees C and analyzed over a period of 21 days. We observed the conversion of the product EPSP into the side product, a cyclic EPSP ketal, and the breakdown product, pyruvate. Thus, the new species reported by solid-state NMR can be accounted for by previously characterized reaction products and side products formed during sample preparation and upon incubation in the solid-state. Our conclusions are also supported by the solution and solid-state NMR studies recently reported [Jakeman et al. (1998) Biochemistry 37, 12012-12019]. These results once again highlight the importance of kinetic competence as a criterion to be used in defining enzyme intermediates and point to the errors in interpretation of results when the time dependence of formation of the proposed intermediates is not considered.     

Chemical trapping of complexes of dihydroxyacetone phosphate with muscle fructose-1,6-bisphosphate aldolase

Dihydroxyacetone phosphate (DHAP) in equilibrium with FDP aldolase of muscle is present in the form of two major covalent complexes. One, representing approximately 60% of total bound substrate, decomposes to Pi and methylglyoxal upon acid denaturation of the enzyme as first reported by Grazi and Trombetta [Grazi, E., & Trombetta, G. (1979) Biochem. J. 175, 361-365]. This is now shown to be the enzyme-eneamine phosphate reaction intermediate since Pi formation is prevented if the acid denaturation is done in the presence of potassium ferricyanide, an oxidant of the eneamine. The enzyme-eneamine aldehyde X Pi 6, presumed to be an intermediate of the slow methylglyoxal synthetase reaction of aldolase, must not be a significant source of the Pi produced upon denaturation and is probably not a significant component of the equilibrium. The oxidation product, the enzyme-imine of phosphopyruvaldehyde, is sufficiently stable in 1 N HCl, t1/2 = 76 min at 0 degree C, to be isolated with the trichloroacetic acid precipitated protein. A second covalent complex, approximately 20-24% of bound dihydroxyacetone [32P]phosphate, remains with the protein during acid denaturation and centrifugation. This acid-stable complex is formed rapidly and is chased rapidly by unlabeled substrate. Its stability in 1 N HCl is similar to that of the ferricyanide-oxidized derivative mentioned above. From this and its reactivity with cyanoborohydride in acid, this complex is thought to be the imine adduct of DHAP with aldolase 4 and/or the carbinolamine complex 3 present in the initial equilibrium. D-Glyceraldehyde 3-phosphate in the carbonyl form also forms an acid-precipitable complex with aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase

The single-turnover kinetic mechanism for the reaction catalyzed by dTDP-glucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-chemical quench mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze quenched samples. The results were compatible with the postulated reaction mechanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose. Next, water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produces the product dTDP-4-keto-6-deoxyglucose. The single-turnover reaction was quenched at various times on the millisecond scale with a mixture of 6 M guanidine hydrochloride and sodium borohydride, which stopped the reaction and reductively stabilized the intermediates and product. Quantitative MALDI-TOF MS analysis of the quenched samples allowed the simultaneous observation of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucose-5,6-ene), and the appearance of product. Kinetic modeling of the process allowed rate constants for most of the steps of the reaction of dTDP-glucose-d(7) to be evaluated. The transient formation and reaction of dTDP-4-ketoglucose could not be observed, because this intermediate did not accumulate to detectable concentrations.     

Mechanism of pyruvate-uridine diphospho-N-acetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate.

The enzyme, phosphoenolpyruvate:uridine-5-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltransferase, which catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine with the liberation of Pi, was found to form a covalent intermediate with the enolpyruvate moiety. Radioactivity from [1-14-C]phosphoenolpyruvate in the forward reaction and from UDP-GlNAc-[1-14-C]enolpyruvate in the reverse reaction was incorporated into the enzyme and remained bound to the protein after precipitation with ammonium sulfate or treatment with sodium dodecyl sulfate and heat. This incorporation from UDP-GlcNAc-[1-14-C]enolpyruvate took place in the absence of Pi. When [32-P,1-14C]phosphoenolpyruvate was used, only 14-C appeared to be incorporated. In the forward reaction, the incorporation was contingent on the removal of UDP-GlcNAc from the transferase. Consistent with the formation of an enzyme-enolpyruvate intermediate, exchange of UDP-[6-3-H]GlcNAc with UDP-GlcNAc-enolpyruvate was observed in the absence of Pi. Nonstoichiometric incorporation of 3H from 3H2O into the product, UDP-GlcNAc-enolpyruvate, was observed and was shown to be due to a product isotope effect. Based on these observations, a mechanism of action for this enzyme is proposed which involves synchronous addition-elimination followed by a second addition-elimination step.     

"Kinetic competence" of the 5-enolpyruvoylshikimate-3-phosphate synthase tetrahedral intermediate

The tetrahedral intermediate formed at the active site of 5-enolpyruvoylshikimate-3-phosphate synthase by reaction of shikimate 3-phosphate with phosphoenolpyruvate was isolated, and its properties in solution and in reaction with enzyme were examined. The intermediate was moderately stable at pH 7.0, with a half-life of 45 min, and showed increasing lifetimes with increasing pH (t1/2 greater than 48 h at pH greater than or equal to 12). The intermediate bound to the enzyme rapidly, with a second order rate constant of 5 x 10(7) M-1 s-1. Upon binding to the enzyme, it reacted to form both products (5-enolpyruvoylshikimate 3-phosphate, Pi) and substrates (shikimate 3-phosphate, phosphoenolpyruvate) in proportions predicted by the rate constants defined previously for reactions occurring at the active enzyme site (Anderson, K.S. Sikorski, J.A., and Johnson, K. A. (1988b) Biochemistry 27, 7395-7406). The kinetics of binding and dissociation of stable phosphonate analogs of the tetrahedral intermediate (Alberg, D., and Bartlett, P.A. (1989) J. Am. Chem. Soc. 111, 2337) were also examined. In comparison to the intermediate, the analogs bound to the enzyme 300-10,000 fold more slowly and at least 300-20,000 times mroe weakly. These results clarify the definitions for kinetic competence of enzyme intermediates and call into question the significance of the slow binding of analogs of transition states or enzyme intermediates.     

Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis

Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed.     

Kinetic competence of enzymic intermediates: fact or fiction?

A number of enzymatic reactions involve intermediates that are not normally released during the reaction. Whether such an intermediate when added to the enzyme reacts as fast or faster than the normal substrates, and thus is "kinetically competent", depends on the degree to which the equilibrium constant for forming the intermediate from the substrates is different on the enzyme surface and in solution, as well as on the relative affinities of the enzyme for substrate and intermediate. Similar values for these equilibrium constants require that the intermediate react slowly, while a far more favorable value for intermediate formation on the enzyme allows the intermediate to react at up to the diffusion-limiting rate. When one intermediate is formed from two substrates, it may react much more rapidly than when two intermediates are formed from two substrates, or one from one. Comparison of the kinetics of the putative intermediate(s) and the substrate(s) can reveal a great deal about the mechanism of the catalytic reaction and the kinetic barrier that normally keeps the intermediate(s) on the enzyme.     

Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin

Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.     

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.     

The pre-steady-state hydrolysis of ATP by porcine brain (Na+ + K+)-dependent ATPase.

The hydrolysis of [gamma-32P]ATP by porcine brain (Na+ + K+)-stimulated ATP phosphohydrolase (EC 3.6.1.3) has been studied at 28 degree C in a rapid mixing quenched-flow apparatus. An "early burst" in the release of Pi from ATP has been observed when the enzyme is mixed with ATP, Na+ and a relatively high concentration of K+ (10 mM) but the burst is less pronounced with 0.5 mM K+. This "early burst" of Pi release is suppressed when the enzyme is pre-mixed with 10 mM K+ or 20% (v/v) dimethylsulphoxide before mixing with ATP and Na+, and premixing of enzyme with Na+ antagonizes this effect of dimethylsulphoxide. The results have been analysed by a non-linear least squares regression treatment and are consistent with a mechanism involving three steps, one of which may be a relatively slow change in enzyme conformation following release of Pi from its covalent linkage with the enzyme, in addition to formation of the enzyme-substrate complex. Rate constants (and S.E.) for these steps have been calculated and the roles of phospho-enzyme and other intermediates in the reaction mechanism of the transport ATPase are dicussed.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

The formation of reactive intermediate(s) of glucose 6-phosphate and lysine capable of rapidly reacting with DNA

Glucose has been shown to react nonenzymatically in vitro with DNA, to form products with spectral properties similar to those observed with the nonenzymatic glycosylation of proteins in vivo. The incubation in vitro of glucose or glucose 6-phosphate with f1 phage DNA results in a time- and concentration-dependent loss of transfection efficiency. It has also been shown that incubation in vitro of pBR322 DNA with glucose 6-phosphate prompts a loss in transformation capability as well as gross DNA alterations. In the present communication, we have investigated a model reaction of glucose 6-phosphate with the amino groups of lysine to form reactive intermediates which are capable of forming covalent adducts with DNA. The preincubation of glucose 6-phosphate and [3H]lysine leads to a time- and concentration-dependent formation of reactive intermediates. These intermediates, which accumulate with time, can subsequently react with single- or double-stranded DNA to form acid-stable complexes. Studies done with synthetic polynucleotides suggest low reactivity of the intermediate with thymidine. The formation of the reactive intermediates is saturated by the addition of excess unlabeled lysine. Once formed the intermediates are insensitive to the addition of aminoguanidine and to reduction by sodium borohydride. The chemical reactions between sugars and lysine reported here and the reactivity of that product with DNA provide a model for exploring the classes of DNA damage that may contribute to the loss of DNA function during aging.     

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.     

Mechanism of action of clostridial glycine reductase: isolation and characterization of a covalent acetyl enzyme intermediate

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-linked binding of Pi is required for continuous steady-state proton-translocating ATP hydrolysis catalyzed by F0.F1 ATP synthase

The presence of medium Pi (half-maximal concentration of 20 microM at pH 8.0) was found to be required for the prevention of the rapid decline in the rate of proton-motive force (pmf)-induced ATP hydrolysis by Fo.F1 ATP synthase in coupled vesicles derived from Paracoccus denitrificans. The initial rate of the reaction was independent of Pi. The apparent affinity of Pi for its "ATPase-protecting" site was strongly decreased with partial uncoupling of the vesicles. Pi did not reactivate ATPase when added after complete time-dependent deactivation during the enzyme turnover. Arsenate and sulfate, which was shown to compete with Pi when Fo.F1 catalyzed oxidative phosphorylation, substituted for Pi as the protectors of ATPase against the turnover-dependent deactivation. Under conditions where the enzyme turnover was not permitted (no ATP was present), Pi was not required for the pmf-induced activation of ATPase, whereas the presence of medium Pi (or sulfate) delayed the spontaneous deactivation of the enzyme which was induced by the membrane de-energization. The data are interpreted to suggest that coupled and uncoupled ATP hydrolysis catalyzed by Fo.F1 ATP synthases proceeds via different intermediates. Pi dissociates after ADP if the coupling membrane is energized (no E.ADP intermediate exists). Pi dissociates before ADP during uncoupled ATP hydrolysis, leaving the E.ADP intermediate which is transformed into the inactive ADP(Mg2+)-inhibited form of the enzyme (latent ATPase).     

5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics

Direct evidence for an enzyme-bound intermediate in the EPSP synthase reaction pathway has been obtained by rapid chemical quench-flow studies. The transient-state kinetic analysis has led to the following complete scheme: (formula; see text) Values for all 12 rate constants were obtained. Substrate trapping experiments in the forward and reverse reactions established the kinetically preferred order of binding and release of substrates and products and showed that shikimate 3-phosphate (S3P) and 5-enolpyruvoylshikimate 3-phosphate (EPSP) dissociate at rates greater than turnover in each direction. Pre-steady-state bursts of product formation were observed in the reaction in each direction indicating a rate-limiting step following catalysis. Single turnover experiments with enzyme in excess over substrate demonstrated the formation of a transient intermediate in both the forward and reverse reactions. In these experiments, the enzymatic reaction was observed by employing a radiolabel in the enol moiety of either phosphoenol pyruvate (PEP) or EPSP. The separation and quantitation of reaction products were accomplished by HPLC monitoring radioactivity. The intermediate was observed as the transient production of radiolabeled pyruvate, formed due to the breakdown of the intermediate in the acid quench used to stop the reaction. The intermediate was observed within 5-10 ms after the substrates were mixed with enzyme and decayed in a reaction paralleling the formation of product in each direction. Thus, the kinetics demonstrate directly the kinetic competence of the presumed intermediate. No pyruvate was formed, on a time scale which is relevant to catalysis, after incubation of the enzyme with dideoxy-S3P and PEP or with EPSP in the absence of phosphate; and so, the intermediate does not accumulate under these conditions. The intermediate broke down to form PEP and EPSP in addition to pyruvate when the reaction was quenched with base rather than acid; therefore, the intermediate must contain the elements of each product. Other experiments were designed to measure directly the phosphate binding rate and further constrain the PEP binding rate. The overall solution equilibrium constant in the forward direction was determined to be 180 by quantitation of radiolabeled reactants and products in equilibrium after incubation with a low enzyme concentration. The internal, active site equilibrium constant was obtained by incubation of radiolabeled S3P with excess enzyme and high concentrations of phosphate and PEP to provide the ratio of [EPSP]/[S3P] = 2.3, which is largely a measure of K4.(ABSTRACT TRUNCATED AT 250 WORDS)