Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.     

Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5'-deoxyadenosyl radical. In most of these enzymes, the 5'-deoxyadenosyl radical is generated by an iron-sulfur cluster-mediated reductive cleavage of S-adenosyl-(S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5'-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Mechanism of inactivation of Escherichia coli aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.     

Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.     

Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol

The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.     

Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.     

Effect of O-methylhydroxylamine on extracellular phage cd]

Study was made of lethal and mutagenic effect of 1 M and 0,5 M O-methylhydroxylamine (OMHA) on extracellular phage Sd. The correlation between chemical changes of the genome and the degree of phage inactivation under the action of OMHA has been established within the range of studied pH (4,5-7,0) of the reaction medium. OMHA in activates the phage at the highest rate at pH 5,0, which agrees with chemical data indicating that the total rate of OMHA modification of cytidine units is maximal at this pH. Inactivation curves of OMHA-treated phage are single-hit at pH investigated, but have a small initial shoulder; at pH 5,0 and 4,5 inactivation curves consist of two exponents, the second exponent having the smallest slope, that is the phage is characterized by an increased resistance to OMHA at this section. The increased phage resistance can be explained by transforming the original product IV (cross-linked with protein) into the product II (N4-methoxy-6-methoxyamine-5,6-dihydrocytidine) which can be repaired in contrast to IV. OMHA has a high mutagenic effect on phage Sd. Under optimal conditions (at pH 4,5) the mutagen induces plaque mutants (up to 6%) among survived phages. The data obtained correlate with the fact that with decreasing pH (from 5,0 to 4,5) the ratio of the "mutagen" unit - N4-methoxycytidine (product III) to the "inactivating" one (product II) increases. The curves of mutation induction under the action of OMHA have a characteristic form with the initial linear section and the maximum or the plateau similar to mutation curves to be observed under the action of radiation and chemical agents.     

Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase).

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.     

Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: characterization of an allylic analogue of a substrate-based radical in the catalytic mechanism

An analogue of lysine, trans-4,5-dehydro-L-lysine (trans-4, 5-dehydrolysine), is a potent inhibitor of lysine 2,3-aminomutase from Clostridium subterminale SB4 that competes with L-lysine for binding to the active site. Inclusion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyridoxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spectrum at g 2.0, which is characteristic of an organic radical. A series of deuterated and (15)N-labeled samples of trans-4,5-dehydrolysine were synthesized and used to generate the EPR signal. Substitution of deuterium for hydrogen at C2, C3, C4, C5, and C6 of trans-4, 5-dehydrolysine led to significant simplifications and narrowing of the EPR signal, showing that the unpaired electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysine. The hyperfine splitting pattern is simplified by use of 4,5-dehydro[3, 3-(2)H(2)]lysine or 4,5-dehydro[4,5-(2)H(2)]lysine, and it is dramatically simplified with 4,5-dehydro-[3,3,4,5,6,6-(2)H(6)]lysine. Spectral simulations show that the EPR signal arises from the allylic radical resulting from the abstraction of a hydrogen atom from C3 of trans-4,5-dehydrolysine. This radical is an allylic analogue of the substrate-related radical in the rearrangement mechanism postulated for this enzyme. The rate constant for formation of the 4,5-dehydrolysyl radical (2 min(-)(1)) matches that for the decrease in the concentration of [4Fe-4S](+), showing that the two processes are coupled. The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methionine takes place with a rate constant of approximately 5 min(-)(1). These kinetic correlations support the hypothesis that radical formation results from a reversible reaction between [4Fe-4S](+) and S-adenosylmethionine at the active site to form [4Fe-4S](2+), the 5'-deoxyadenosyl radical, and methionine as intermediates.     

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12.

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.     

Suicide inactivation of fructose-1,6-bisphosphate aldolase

2-Keto-4,4,4-trifluorobutyl phosphate (HTFP) was prepared from 3,3,3-trifluoropropionic acid. HTFP acts as an irreversible inhibitor of rabbit muscle aldolase: the loss of activity was time dependent and the inactivation followed a pseudo-first-order process. Values of 1.4 mM for the dissociation constant and 2.3 X 10(-2) s-1 for the reaction rate constant were determined. The kinetic constants do not depend on the enzyme concentration. No effect of thiols on the inactivation rate was detected. Only 1-2 mol of fluoride ions was liberated per inactivated subunit, indicative of a low partition ratio. Dihydroxyacetone phosphate protected the enzyme against the inactivation in a competitive manner, and glyceraldehyde 3-phosphate protected as if it formed a condensation product with HTPF. 5,5'-Dithiobis(2-nitrobenzoic acid) thiol titration showed the loss of one very reactive thiol group per enzyme subunit after inactivation. All those observations seem to agree with a suicide substrate inactivation of aldolase by HTPF.     

Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.     

Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation

Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.     

Inactivation of dihydropyrimidine dehydrogenase by 5-iodouracil

5-Iodouracil was a substrate for bovine liver dihydropyrimidine dehydrogenase (DHPDHase) and was a potent inactivator of the enzyme. NADPH increased the rate of inactivation and thymine protected against inactivation. These findings suggest that 5-iodouracil was a mechanism-based inactivator. However, dithiothreitol and excess 5-iodouracil protected the enzyme against inactivation. Thus, a reactive product, presumably 5-iodo-5,6-dihydrouracil generated through the enzymatic reduction of 5-iodouracil, was released from DHPDHase during processing of 5-iodouracil. Since only 18% of [6-3H]5-iodouracil reduced by DHPDHase was covalently bound to the enzyme and radiolabel was not lost to the solvent as tritium, the partition coefficient for inactivation was 4.5. However, the enzymatic activity was completely titrated with 1.7 mol of 5-iodouracil per mol of enzyme-bound flavin. These results indicate that there was 0.31 mol of enzyme-bound inactivator per mol of enzyme flavin. This suggests there were 3.2 flavins per active site, which is consistent with the report of multiple flavins per enzymic subunit (Podschun, B., Wahler, G., and Schnackerz, K. D. (1989) Eur. J. Biochem. 185, 219-224). DHPDHase was inactivated by 2.1 mol of racemic 5-iodo-5,6-dihydrouracil per mol of active sites. The stoichiometry for inactivation of the enzyme by the nonenzymatically generated enantiomer of 5-iodo-5,6-dihydrouracil was calculated to be 1. Two radiolabeled fragments were isolated from a tryptic digest of DHPDHase inactivated with radiolabeled 5-iodouracil. The amino acid sequences of these peptides were Asn-Leu-Ser-X-Pro-His and Asn-Leu-Ser-X-Pro-His-Gly-Met-Gly-Glu-Arg where X was the modified amino acid containing radiolabel from [6-3H]5-iodouracil. Fast atom bombardment mass spectral analysis of the smaller peptide yielded a protonated parent ion mass of 782 daltons that was consistent with X being a S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteinyl residue.     

The radiolysis of glyceraldehyde-3-phosphate dehydrogenase.

The yields in molecules per 100 eV for active-site and sulphydryl loss from glyceraldehyde-3-phosphate dehydrogenase have been determined in nitrous-oxide-saturated, aerated and argon-saturated solutions. Molecular hydrogen peroxide produces a sulphenic acid product, which can be repaired by post-irradiation treatment with dithiothreitol. Comparison of the yields under various conditions showed that in aerated solutions both .OH and .O2-radicals inactivated the enzyme with an efficiency of about 26 per cent. However, the efficiency of .OH in air-free solutions was less, and inactivation by .H and eaq- did not appear to be appreciable. There is a correlation between SH loss and loss of active sites.     

Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin?

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.     

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.