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.     

Stoichiometry and stability of the adduct formed between human 4-aminobutyrate aminotransferase and 4-aminohex-5-enoate: sequence of a labelled peptide

The reaction between human 4-aminobutyrate aminotransferase and the anti-epileptic drug 4-aminohex-5-enoate, an irreversible inhibitor of the enzyme, has been studied using the radiolabelled compound. The inactivated enzyme was found to lose radiolabel over a period of a few days at 37 degrees C but even in the presence of the coenzyme, pyridoxal phosphate, no enzyme activity returned. At 4 degrees C the radiolabelled inhibitor remained stably bound. The amount of enzyme-bound 4-aminohex-5-enoate was significantly less than would be expected if one mol of inhibitor was bound per mol of active site. Reversed phase chromatography of a tryptic digest of the labelled enzyme showed that, apart from material eluting at the front of the chromatogram, all of the radioactivity was in a single fraction. This fraction contained a peptide, the sequence of which indicated that it included the lysine that binds the coenzyme and that the major release of radioactivity occurred in an Edman degradation cycle corresponding to this residue.     

L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site

A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

Demonstration that lysine-501 of the alpha polypeptide of native sodium and potassium ion activated adenosinetriphosphatase is located on its cytoplasmic surface

Evidence that the peptide HLLVMKGAPER, which can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion, is located on the cytoplasmic surface of the native enzyme has been obtained. An immunoadsorbent directed against the carboxy-terminal sequence of this tryptic peptide has been constructed. The peptide KGAPER was synthesized by solid-phase techniques. Antibodies against the sequence -GAPER were purified by immunoadsorption, using the synthetic peptide attached to agarose beads. These antibodies, in turn, were coupled to agarose beads to produce an immunoadsorbent. Sealed, right-side-out vesicles, prepared from canine kidneys, were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin, respectively. A tryptic digest of these labeled vesicles was passed over the immunoadsorbent. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digests obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction as applied to these vesicles, it could be concluded that this increase in incorporation resulted only from the access that the reagents gained to the inside of the vesicles in the presence of saponin and that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.     

Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase

Smooth muscle myosin light chain kinase, purified to homogeneity, has a molecular weight of 130,000 +/- 5,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme has a specific activity under maximal conditions of 30 mumol Pi transferred to myosin light chain/mg kinase/min at 24 C and is totally dependent on calmodulin and calcium for activity. Incubation of myosin kinase with the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase results in the covalent incorporation of up to one mol of phosphate per mol of myosin kinase in the absence of bound calmodulin. Limited tryptic digestion of the radioactively labeled kinase indicates that all of the label has been incorporated into a single tryptic peptide (mol wt approximately 22,000), suggesting that a single site is being phosphorylated. Phosphorylation of myosin kinase lowers the rate at which the kinase phosphorylates myosin light chain. The lower rate of light chain phosphorylation is due to a weaker binding of calmodulin to the phosphorylated kinase than to the unphosphorylated kinase. Cyclic adenosine 3':5'-monophosphate-dependent phosphorylation of the kinase actin-myosin interaction represents a possible link between hormonal binding to smooth muscle receptors and muscle relaxation. A scheme for this sequence of events is presented.     

The NADP+-binding site of ferredoxin-NADP+ reductase. Sequence of the peptide containing the essential lysine residue

The flavoprotein ferredoxin-NADP+ reductase is inactivated and loses its ability to bind NADP+ during covalent modification of a lysine by 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, 14-24]. The substrate NADP+ gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl-lysine which was isolated by high-performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP+. The sequence of the dansyl-peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl-peptide. Thus, the octapeptide containing the essential lysine residue has the following sequence: H2N-Ser-Val-Ser-Leu-Cys-Val-Lys-Arg-COOH. A comparison with corresponding sequences of other known NADP+-dependent dehydrogenases is attempted.     

Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

Identification of a lysine residue at a nucleotide binding site in the firefly luciferase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine

Firefly luciferase is 80-90% inactivated within 3 h upon incubation with the adenine nucleotide analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). Although 4 mol of 14C-FSBA/mol of enzyme is irreversibly bound during inactivation, only 1 mol of 14C-FSBA appears to be specifically directed to an adenine nucleotide binding site on the enzyme. The other 3 mol of 14C-FSBA is bound to the protein nonspecifically. The major radioactive peptide in a tryptic digest os labeled luciferase was isolated and shown to have the following amino acid sequence: *Lys-Gly-Glx-Asx-Ser-Lys, where *Lys is the radioactive derivative of the lysine residue that was sulfonylated during the inactivation.     

Acylation of aspartate aminotransferase

1. Acetylation of aspartate aminotransferase from pig heart inhibits completely the enzymic activity when the coenzyme is in the amino form (pyridoxamine phosphate) or when the coenzyme has been removed, but not when the coenzyme is in the aldehyde form (pyridoxal phosphate). 2. The group the acylation of which is responsible for the inhibition has been identified with the in-amino group of a lysine residue at the coenzyme-binding site. Moreover, in the pyridoxamine-enzyme the amino group of the coenzyme is also acetylated. 3. The reactivity of the coenzyme-binding lysine residue is greatly different in the pyridoxamine-enzyme and in the apoenzyme, suggesting the possibility of an interaction of its in-amino group with pyridoxamine or with other groups on the protein.     

Sequence of a tryptic peptide from the NADPH binding site of the enoyl reductase domain of fatty acid synthase

Fatty acid synthase from the uropygial gland of goose was inhibited by treatment with pyridoxal 5'-phosphate by selectively modifying a lysine residue at the NADPH binding site of the enoyl reductase domain (A. J. Poulose and P. E. Kolattukudy (1980) Arch. Biochem. Biophys. 201, 313-321). Distribution of radioactivity in tryptic peptides generated from the synthase treated with pyridoxal 5'-phosphate/NaB3H4 in the presence and absence of 2'-monophosphoadenosine-5'-diphosphoribose, which protects the enzyme from inactivation by pyridoxal phosphate, showed that modification of one specific peptide was prevented by the protector. This peptide was purified by a combination of Sephadex G-25 column chromatography, anion-exchange chromatography, and high-performance liquid chromatography. The primary structure of this peptide is Val-Phe-Thr-Thr-Val-Gly-Ser-Ala-Glu-Lys(Pxy)-Arg.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil.

Uracil analogues with appropriate substituents at the 5-position inactivated dihydropyrimidine dehydrogenase (DHPDHase). The efficiency of these inactivators was highly dependent on the size of the 5-substituent. For example, 5-ethynyluracil inactivated DHPDHase with an efficiency (kinact/Ki) that was 500-fold greater than that for 5-propynyluracil. 5-Ethynyluracil inactivated DHPDHase by initially forming a reversible complex with a Ki of 1.6 +/- 0.2 microM. This initial complex yielded inactivated enzyme with a rate constant of 20 +/- 2 min-1 (kinact). Thymine competitively decreased the apparent rate constant for inactivation of DHPDHase by 5-ethynyluracil. The absorbance spectrum of 5-ethylnyluracil-inactivated DHPDHase was different from that of reduced enzyme. These optical changes were correlated with the loss of enzymatic activity. 5-Ethynyluracil inactivated DHPDHase with a stoichiometry of 0.9 mol of inactivator per mol of active site. Enzyme inactivated with [2-14C]5-ethynyluracil retained all of the radiolabel after denaturation in 8 M urea, but lost radiolabel under acidic conditions. These results suggested that inactivation was due to covalent modification of an amino acid residue and not due to modification of a noncovalently bound prosthetic group. A radiolabeled peptide was isolated from a tryptic digest of the enzyme inactivated with [2-14C]5-ethynyluracil. The sequence of this peptide was Lys-Ala-Glu-Ala-Ser-Gly-Ala-Y-Ala-Leu-Glu-Leu-Asn-Leu-Ser-X-Pro-His-Gly- Met-Gly-Glu-Arg, where X and Y were unidentified amino acids. Since the radiolabel was lost from the peptide during the first cycle on the amino acid sequenator, the position of the radiolabeled amino acid was not determined. The amino acid residue designated by X was identified as a cysteine from previous work with DHPDHase inactivated with 5-iodouracil. In contrast to 5-ethynyluracil, 5-cyanouracil was a reversible inactivator of the enzyme. 5-Cyanouracil-inactivated enzyme slowly regained activity (t1/2 = 1.8 min) after dilution into the standard assay. DHPDHases isolated from rat, mouse, and human liver had similar sensitivities to inactivation by 5-alkynyluracils.     

The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.     

Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction.

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.     

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.