Studies on the biosynthesis of cyclitols, XXXV. On the mechanism of action of myo-inositol-1-phosphate synthase from rat testicles.

The animal myo-inositol-1-phosphate synthase is competitively inhibited by pyridoxal phosphate and trinitrobenzensulphonate, both compounds known to prevent Schiff's base formation. When incubated with labelled substrate and then treated with NaBH4, label can be recovered in the enzyme protein. In analogous experiments with tritiated NaBH4 the enzyme protein also becomes labelled; after hydrolysis of such protein only one labelled compound, derived from lysine and D-glucose 6-phosphate, could be isolated. Its exact structure is not yet known. From these results it can be concluded that during its action myo-inositol-1-phosphate synthase forms a Schiff's base with its substrate, in analogy to the class I aldolases.     

Mechanism of action of uridine diphosphoglucose dehydrogenase. Evidence for a second reversible dehydrogenation step involving an essential thiol group.

Although the enzyme UDP-glucose dehydrogenase from beef liver (E.C. 1.1.1.22) is known to abstract the pro-R hydrogen stereospecifically at carbon 6 of the glucose moiety of the substrate by a reversible step in converting UDP-glucose to UDP-alpha-D-gluco-hexodialdose (UDP-Glc-6-CHO), prolonged incubation of the enzyme with UDP-glucose and tritium-labeled NADH, under conditions favoring hydrogen exchange between the two, results in equivalent labeling of both hydrogens at carbon 6. This shows that the pro-S hydrogen at carbon 6 is also abstracted by a reversible process which must then involve a derivative of the carboxyl group of UDP-glucuronic acid (UDP-GlcUA) that is capable of reversible hydrogenation-dehydrogenation. It is the hydrolysis of this derivative that accounts for the well known irreversibility of the overall reaction. Derivatization of the enzyme's essential thiol group with 5,5'-dithiobis-(2-nitrobenzoate) eliminates the ability of the enzyme to either oxidize or reduce UDP-Glc-6-CHO. Replacement of the 5-thio-2-nitrobenzoate group with cyanide fully restores the enzyme's capacity to reduce UDP-Glc-6-CHO but has no effect on the inhibition of the oxidation to UDP-GlcUA. This indicates that the essential thiol group is involved in the second reversible dehydrogenation step and serves to form a thiol ester with the carboxyl of the product, UDP-GlcUA. It is suggested that thiol ester intermediates are a general characteristic of all 4-electron NAD-linked dehydrogenase reactions.     

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.     

Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP+ oxidoreductase after affinity labeling with periodate-oxidized NADP+

Spinach ferredoxin-NADP+ oxidoreductase was inactivated by treatment with 2',3'-dialdehyde NADP+ (periodate-oxidized NADP+), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP+ and NaB3H4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys*-Met-Tyr-Ile-Gln-Thr-Arg, where Lys* represents the derivatized lysine. The sequence obtained corresponds to residues 242-250 in the primary structure of spinach ferredoxin-NADP+ reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576-6583].     

Inactivation of the dadB Salmonella typhimurium alanine racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism

The pyridoxal phosphate dependent Salmonella typhimurium dadB alanine racemase was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and O-acetyl-D-serine. Enzyme inactivation with each isomer of beta-chloro[14C]alanine followed by NaBH4 reduction and trypsin digestion afforded a single radiolabeled peptide. In the same manner, NaB3H4-reduced native enzyme gave a single labeled peptide after trypsin digestion. Purification and sequencing of these three radioactive peptides revealed them to be a common, unique hexadecapeptide which contained labeled lysine at position 6 in each case. Enzyme which had been inactivated, but not reductively stabilized with NaBH4, released a labile pyridoxal phosphate-inactivator adduct on denaturation. The structure of this adduct suggests that the enzyme was inactivated by trapping the coenzyme in a ternary adduct with inactivator and the active site lysine. Under denaturing conditions, facile alpha,beta-elimination occurred, releasing the aldol adduct of pyruvate and pyridoxal phosphate. Reduction of the ternary enzyme adduct blocked this elimination pathway. The overall mechanism of racemase inactivation is discussed in light of these results.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.     

Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.

S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.     

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.     

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.     

Active site residues and mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-2H]-d-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of kcat is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-2H]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.     

The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram.

1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH.     

Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida utilis.

1. Periodate-oxidized NADP+ inhibits the catalytic activity of glucose 6-phosphate dehydrogenase from Candida utilis, competing with NADP+. 2. Incubation of the enzyme with the coenzyme analogue causes partial reversible inactivation of the enzyme as a result of affinity labelling of the coenzyme-binding site. 3. Some kinetic values of the reaction were calculated. 4. The inactivation can be made irreversible by treatment with NaBH4, which reduces a Schiff base formed between an aldehyde group on the coenzyme analogue and a lysine residue on the enzyme. 5. Complete inactivation can be correlated with the binding of only one inhibitor to each enzyme subunit. 6. The lysine residue involved in the binding of the inhibitor is present at the coenzyme-binding site.     

Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride

Further chemical evidence has been obtained using NaB3H4 to support our previous assignment of a thiol ester bond in human C3 (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768). Following trypsin activation of human C3 in the presence of NaB3H4, 3H was shown to have incorporated specifically into the alpha'-chain of C3b. Subsequent fragmentation of [3H]C3b with porcine elastase further localized the label to the C3d subdomain. Under identical conditions, native C3 or C3 pretreated with trypsin (C3b) showed low reactivity with NaB3H4. A tryptic peptide containing the 3H label was isolated following digestion of [3H]C3b on activated thiol-Sepharose. After hydrolysis and saponification of the peptide hydrolysate, amino acid analysis indicated that the 3H had been incorporated into alpha-amino-delta-hydroxyvaleric acid, the product expected from reduction of an ester bond involving a glutamyl residue. On sequence analysis of the labeled peptide, the 3H was shown to reside at the position of the glutamyl residue previously proposed to be involved in the thiol ester bond. The residue at this position was confirmed as alpha-amino-delta-[3H] hydroxyvaleric acid by high performance liquid chromatography analysis and, after back hydrolysis, by amino acid analysis. These data significantly strengthen earlier studies which indicated the presence of a beta-Cys-gamma-Glu thiol ester bond in human C3.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photomodification of a serine at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase by vanadate

Irradiation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach in the presence of vanadate at 4 degrees C resulted in rapid loss of carboxylase activity. The inactivation was light and vanadate dependent. When the enzyme was irradiated in the presence of the substrate ribulose 1,5-bisphosphate or an analogue such as fructose 1,6-bisphosphate, the inactivation was greatly reduced. Sodium bicarbonate and phosphate also protected against inactivation. No additional protection was observed in the presence of Mg2+ nor did Mg2+ alone protect. Carboxylase activity could be partially restored by treatment with NaBH4, and the photomodified protein could be tritiated with NaB3H4. Amino acid analysis showed that the tritium had been incorporated into serine. The data suggest that an active-site serine is photooxidized by vanadate to an aldehyde which results in activity loss. Irradiation in the presence of vanadate also resulted in cleavage in the large subunit of the enzyme which was subsequent to inactivation.     

Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

Phospholipase C from Bacillus cereus. Evidence for essential lysine residues.

1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.     

Modification of pig M4 lactate dehydrogenase by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.