Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by 13C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using [2-13C]PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP (152 ppm) and EPSP bound to the enzyme in a ternary complex with phosphate (161 ppm). These peak assignments account for our quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted; the intermediate could be isolated by quenching with either triethylamine or 0.2 N KOH, allowing direct quantitation of the species bound to the enzyme. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal [Leo et al. (1990) J. Am. Chem. Soc. (in press)]. The rate of formation of the EPSP ketal was very slow, 3.3 X 10(-5) s-1, establishing that it is a side product of the normal enzymatic reaction, probably arising as a breakdown product of the tetrahedral intermediate. A slow formation of pyruvate was also observed and is attributable to the enzymatic hydrolysis of EPSP, with 5% of the enzyme sites occupied by EPSP and hydrolyzing EPSP at a rate of 4.7 X 10(-4) s-1. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, an NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

"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.     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A

Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.     

Hydrolysis of human high-molecular-mass kininogen by human plasma kallikrein. Proposal of a new model concept for the course of reaction in presence and absence of C1(-)-inhibitor

Hydrolysis of high-molecular-mass kininogen was studied by following the changes in the amounts of substrate, intermediates and products as a function of time using quantitative polyacrylamide-gel electrophoresis (silver staining). The experimental data was analysed on the basis of the concept that the overall reaction is composed of three hydrolysis reactions, two positional-change processes of intermediates at the active site, and two product-substrate exchange processes. It is proposed C1(-)-inhibitor to form two types of complexes with kallikrein, one with non-covalent and one with covalent bonds. With an adequately chosen set of reaction-partner concentrations and four different kinds of experimental conditions with respect to kininogen and inhibitor addition to kallikrein, the following results were obtained: 1) Non-covalently bound inhibitor has no effect on the first and the second hydrolysis reaction, but efficiently interferes with the third hydrolysis reaction; 2) Nicked kininogen (first intermediate; one of the two bradykinin bonds split) for the second bond to be hydrolysed undergoes a positional change during which it remains strongly bound to the enzyme, never exchanges with kininogen, and is not displaced by non-covalently bound inhibitor; 3) Intermediate kinin-free kininogen (second intermediate; both bradykinin bonds split and bradykinin released) prior to turning over into stable kinin-free kininogen (final product; histidine-rich fragment split off and released) undergoes a positional change involving dissociation and reassociation so that non-covalently bound inhibitor finds access to the active site; 4) Intermediate kinin-free kininogen to sustain multiple turnovers exchanges with kininogen via a stable complex of such structure that during this process non-covalently bound inhibitor cannot or can only slightly interfere; 5) Stable kinin-free kininogen to sustain multiple turnovers exchanges with intermediate kinin-free kininogen via free enzyme with the effect that non-covalently bound inhibitor efficiently interferes; 6) As hydrolysis proceeds more and more inhibitor becomes covalently bound, gradually leading to complete inactivation of the enzyme.     

Chemistry of the inactivation of cytosolic aspartate aminotransferase by serine O-sulfate

The reaction of serine O-sulfate with cytosolic aspartate aminotransferase [John, R.A., & Fasella, P. (1969) Biochemistry 8, 4477] has been reinvestigated. As in the corresponding reaction with beta-chloroalanine [Morino, Y., Osman, A.M., & Okamoto, M. (1974) J. Biol. Chem. 249, 6684], the enzyme is inactivated over a 10-min period, and the absorption maximum at pH 5.4 shifts from 430 to 336 nm. Upon prolonged standing the peak shifts again over a period of 20 h to 455 nm, a behavior entirely similar to that reported by Morino et al. for beta-chloroalanine in the presence of 3 M formate. When the pH of either the 10-min product (1a) or the 20-h product (1b) is raised to 11 or above, a yellow, diffusible compound (2) is released from the protein. This compound as well as its dephosphorylation and reduction products has been isolated and studied by NMR spectroscopy. Compound 2 is identical with a compound formed from serine sulfate and glutamate decarboxylase by a similar reaction sequence [Likos, J.J., Ueno, H., Feldhaus, R.W., & Metzler, D.E. (1982) Biochemistry (preceding paper in this issue)] and is the product of an aldol condensation of pyruvate with pyridoxal phosphate. When the 20-h product 1b is reduced with sodium borohydride and then heated in a boiling water bath, a material identical with the reduction product of 2 is released. We propose that the 20-h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many enzyme-activated inhibitors of pyridoxal phosphate dependent enzymes.     

Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis

In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.     

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.     

Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions.

By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.     

13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.     

Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.     

Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli. Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beechem, J. M., and Matthews, C. R. (1995) Biochemistry 34, 1867-1877], the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques. Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A. The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s). Viewed within the context of the kinetic folding model of alphaTS [Bilsel, O., Zitzewitz, J. A., Bowers, K. E. , and Matthews, C. R. (1999) Biochemistry 38, 1018-1029], the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit. The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure.     

Structure of the sodium borohydride-reduced N-(cyclopropyl)glycine adduct of the flavoenzyme monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.     

Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that tryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with beta-mercaptoethanol in competition with hydride transfer from H(4)folate to form dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate concentration. This observation seemed inconsistent with the conclusion that both products arose from a common intermediate in which CH(2)H(4)folate was already bound to the enzyme. The enigma was resolved by a kinetic model that allowed for differential partitioning of the intermediate formed on each of the two subunits of the homodimeric enzyme in forming the two different products. With three C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit, but was inhibited when both subunits were occupied. With all mutants, dTMP formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enigma of the cofactor-dependent product distribution and supports the conclusion that the exocyclic methylene intermediate is common to both HETM-dUMP and dTMP formation.     

Correlation of copper valency with product formation in single turnovers of dopamine beta-monooxygenase

Chemical- and freeze-quench EPR techniques have allowed single-turnover studies of the copper-containing enzyme dopamine beta-monooxygenase. Reduction of enzyme by a stoichiometric amount of ascorbate followed by rapid mixing with tyramine leads to oxidation of bound copper and formation of hydroxylated product in the expected 2:1 ratio. The tyramine dependence of single turnovers yields a limiting rate of 82 +/- 9 s-1 and Km of 3 +/- 1 mM, in agreement with kinetic modeling based on steady-state parameters. Together these results show that the reduced enzyme is a catalytically competent species, with bound copper acting as the sole reservoir of reducing equivalents. The correlation of copper oxidation and substrate hydroxylation rules out significant antiferromagnetic spin coupling in the enzyme-product complex. Since the enzyme-product complex's Cu2+ EPR signal is absent in the transient approach to the steady state [Brenner, M. C., Murray, C. J., & Klinman, J. P. (1989) Biochemistry (preceding paper in this issue)], this result implies that ascorbate reduces copper in the enzyme-product complex. These findings have important consequences for catalysis and active site structure in dopamine beta-monooxygenase.     

Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: partitioning of reaction intermediates in D2O and activation by phosphite dianion

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.     

The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction

Spinach-leaf ribulose-5-phosphate kinase catalyzes the reaction of (Rp)-[beta, gamma-18O, gamma-18O]adenosine 5'-(3-thiotriphosphate) with ribulose 5-phosphate to form ribulose 1-[18O]phosphorothioate 5-phosphate. This product is incubated with CO2, Mg2+, and ribulose-bisphosphate carboxylase to form the [18O]phosphorothioate of D-glycerate. Reduction of this material using phosphoglycerate kinase/ATP, glyceraldehyde-3-phosphate dehydrogenase/NADH, triose-phosphate isomerase, and glycerol-phosphate dehydrogenase/NADH produces glycerol 3-[18O]phosphorothioate, which is subjected to ring closure using diethylphosphorochloridate. This in-line reaction produces a diastereoisomeric mixture of glycerol 2,3-cyclic phosphorothioates. 31P NMR spectroscopy was used to analyze the 18O content of the products. The anti-diastereoisomer, which is the major isomer formed and corresponds to the downfield 31P NMR signal (Pliura, D.H., Schomburg, D., Richard, J.P., Frey, P.A., and Knowles, J.R. (1980) Biochemistry 19, 325-329), retains the 18O label. This observation indicates that the ribulose-5-phosphate kinase reaction proceeds with inversion of configuration at phosphorus. The reaction is, therefore, unlikely to involve the participation of a covalent phosphoryl-enzyme intermediate.