Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.

Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6R)-5,10-methenyltetrahydropteroylpolyglutamate believed to be (6R,11R)-5,10-hydroxymethylenetetrahydropteroylpolyglutamate , a putative intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V. (1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates and the role of glycine in the reaction was addressed. Evidence suggests that (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is a catalytically competent intermediate in the enzyme-catalyzed hydrolysis of (6R)-5,10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 microM for (6R)-5,10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is below 0.5 microM. The kcat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbing at 502 nm which is formed from glycine and (6S)-5-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5,10-methenyltetrahydropteroyltetraglutamate, suggesting that the enzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H]glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity in mechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamates.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.     

The nonenzymatic hydrolysis of oligoribonucleotides. VII. Structural elements affecting hydrolysis

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.     

Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation.

The inactivation of glycosidases by 2-deoxy-2-fluoroglycosides has been shown previously to occur via the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate [Withers, S. G., & Street, I. P. (1988) J. Am. Chem. Soc. 110, 8551]. Further characterization of this process with Agrobacterium beta-glucosidase is described, and the range of glycosides engaging in this behavior is examined. Inactivation is shown to be accompanied by the release of a stoichiometric "burst" of aglycon, thereby providing a new class of active site titration agents for glycosidases. The rate of inactivation is shown to be very strongly dependent on the leaving group ability of the aglycon, the slowest inactivator studied (p-nitrophenyl2-deoxy-2-fluoro-beta-D-glucopyranoside) yielding only partial inactivation due to turnover of the intermediate becoming competitive with its formation. Such turnover of the intermediate is shown to be greatly accelerated by transglycosylation to a suitable glycoside bound in the aglycon site, resulting in the release of a disaccharide product which was isolated and characterized. The pH dependences of both the formation and the hydrolysis of the 2-deoxy-2-fluoroglycosyl-enzyme closely resemble those of each step for normal catalysis, indicating that the same catalytic groups are involved in both processes. A model system for the partial "steady-state" inactivation observed previously [Withers, S. G., Rupitz, K., & Street, I. P. (1988) J. Biol. Chem. 263, 7929] with certain other glycosidases was established by incubating the enzyme with an inactivator known to undergo relatively rapid transglycosylation in the presence of various concentrations of a suitable reactivator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen

The possibility of the existence of a covalent enzyme-phosphoryl intermediate, E-PO3, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase (PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22% of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be rate-limiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 32PO3 group of [gamma-32P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has been determined over the range pH 3-8; a feature with a pKa of approximately 6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a two-stem pseudo Uni Bi mechanism that utilizes a covalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.     

Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction.

The enzyme catalysing the reduction of adenosine 5'-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 degrees C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2-3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5'-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 degrees C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2-3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2-3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.     

Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate.

A recombinant protein-tyrosine-phosphatase has been expressed in Escherichia coli and purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using affinity chromatography. When the phosphatase was allowed to react with 32P-labeled substrates and then rapidly denaturated, a 32P-labeled phosphoprotein could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transient formation of a 32P-labeled phosphoprotein was observed, and the 32P-labeled protein disappeared as substrate was consumed. In the presence of 32P-labeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosine-phosphatase. Site-directed mutagenesis of a catalytically essential cystine residue (position 215) in the recombinant protein resulted in an inactive enzyme, and no phosphoprotein was formed. The 32P-labeled phosphoprotein showed a maximum lability between pH 2.5 and 3.5 and was rapidly decomposed in the presence of iodine. These properties, along with additional site-directed mutations, suggest that the protein-tyrosine-phosphatase forms a covalent thiol phosphate linkage between Cys215 and phosphate.     

Evidence from chemical degradation studies for a covalent bond from 5-fluoro-2'-deoxyuridylate to N-5 of tetrahydrofolate in the ternary complex of thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate

In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Structure of a transient intermediate for GTP hydrolysis by ras

The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.     

Evidence for a pro-oxidant intermediate in the assembly of cytochrome oxidase

The hydrogen peroxide sensitivity of cells lacking two proteins, Sco1 and Cox11, important in the assembly of cytochrome c oxidase (CcO), is shown to arise from the transient accumulation of a pro-oxidant heme A-Cox1 stalled intermediate. The peroxide sensitivity of these cells is abrogated by a reduction in either Cox1 expression or heme A formation but exacerbated by either enhanced Cox1 expression or heme A production arising from overexpression of COX15. Sco1 and Cox11 are implicated in the formation of the Cu(A) and Cu(B) sites of CcO, respectively. The respective wild-type genes suppress the peroxide sensitivities of sco1Delta and cox11Delta cells, but no cross-complementation is seen with noncognate genes. Copper-binding mutant alleles of Sco1 and Cox11 that are nonfunctional in promoting the assembly of CcO are functional in suppressing the peroxide sensitivity of their respective null mutants. Likewise, human Sco1 that is nonfunctional in yeast CcO assembly is able to suppress the peroxide sensitivity of yeast sco1Delta cells. Thus, a disconnect exists between the respiratory capacity of cells and hydrogen peroxide sensitivity. Hydrogen peroxide sensitivity of sco1Delta and cox11Delta cells is abrogated by overexpression of a novel mitochondrial ATPase Afg1 that promotes the degradation of CcO mitochondrially encoded subunits. Studies on the hydrogen peroxide sensitivity in CcO assembly mutants reveal new aspects of the CcO assembly process.     

Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate.

The rabbit liver enzymes 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, and 10-formyltetrahydrofolate synthetase have been purified to apparent homogeneity. Polyacrylamide gel electrophoresis patterns suggest a single protein is responsible for all three catalytic activities. The properties of the dehydrogenase and cyclohydrolase activities suggest that a single active site may catalyze these two reactions. This conclusion is based on spectral changes observed in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate, the similarity of dissociation constants determined from initial velocity studies for the two reactions, and the similarity of the pH-activity curves for the two reactions. NADP⁺ and NADPH lower the K_m for 5,10-methenyltetrahydrofolate 2- to 3-fold above pH 7 in the cyclohydrolase reaction but below pH 7 they act as partial inhibitors.