Essential Lysyl Residues Research Articles

Evidence for an essential lysyl residue in phospholipase D from Streptomyces sp. by modification with diethyl pyrocarbonate and pyridoxal 5-phosphate.

Diethyl pyrocarbonate inactivated phospholipase D from Streptomyces PMF with second-order rate constants of 0.7 M-1 s-1 at pH 6.1 or 222 M-1 s-1 at pH 8.3 and 25 degrees C, and modified 5 His residues per enzyme molecule. The His residues, however, were not essential for activity because: (a) the second-order rate constants for reaction of diethyl pyrocarbonate with the His residues of the enzyme, which were 1.4 M-1 s-1 at pH 6.1 or 7.2 M-1 s-1 at pH 8.3 and 25 degrees C, differed, both at low and high pH values, from the inactivation rates, and (b) the reversal of His modification by hydroxylamine was not accompanied by recovery of activity. As demonstrated by dinitrophenylation experiments carried out on the treated enzyme, diethyl pyrocarbonate also modified up to 20 Lys residues per enzyme molecule. Other amino acid residues and the conformation and hydrodynamic volume of the enzyme were not modified. The involvement of a Lys residue in enzyme activity was confirmed through experiments with pyridoxal 5-phosphate which inactivated phospholipase D, after NaBH4 reduction, with a second-order rate constant of 3.5 M-1 s-1 at pH 8.5 and 15 degrees C. The inactivation took place with concomitant modification of 4 Lys residues, only one of which was found to be essential using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1538). Dicaproyl phosphatidylcholine markedly protected the enzyme against inactivation by DEP or PLP, and this strongly suggests that the essential Lys residue is located in or near the substrate binding site.

An Essential Lysyl Residue (Lys208) in the Substrate‐Binding Site of Porcine FAD‐Containing Monooxygenase

The substrate (amine)‐binding site of porcine FAD‐containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5′‐phosphate (pyridoxal‐P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal‐P. Upon reduction of pyridoxal‐P ‐treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal‐P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal‐P ‐treated FMOs indicated that the lysyl residues were modified by pyridoxal‐P. The about 74% inactivation of the enzymic activity on covalent pyridoxal‐P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N ‐dimethylaniline. The FMO covalently modified with pyridoxal‐P in the presence or absence of N,N ‐dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse‐phase high‐performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal‐P and was detected at 325 nm in the absence of N,N ‐dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186–208.These results suggest that Lys208 plays an important role in the substrate (amine)‐binding site of FMO.

An essential lysyl residue (Lys208) in the substrate-binding site of porcine FAD-containing monooxygenase.

The substrate (amine)-binding site of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5'-phosphate (pyridoxal-P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal-P. Upon reduction of pyridoxal-P-treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal-P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal-P-treated FMOs indicated that the lysyl residues were modified by pyridoxal-P. The about 74% inactivation of the enzymic activity on covalent pyridoxal-P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N-dimethylaniline. The FMO covalently modified with pyridoxal-P in the presence or absence of N,N-dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse-phase high-performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal-P and was detected at 325 nm in the absence of N,N-dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186-208. These results suggest that Lys208 plays an important role in the substrate (amine)-binding site of FMO.

Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase

Retinal oxidase, which synthesizes all- trans and 9-cis retinoic acid from all- trans and 9- cis-retinal, has been purified from rabbit cyter pyrosol. The substrate-binding site of the retinal oxidase was investigated with some chemical modification reagents. Lysyl-specific pyridoxal 5′-phosphate (PLY') and cysteinyl-specific p-chloromercuribenzoate (PCMB) competitively inhibited the activity of the retinal oxidase, and the inhibition could be prevented by the presence of all- trans-retinal or its derivatives. Treatment with sodium borohydride (NaBH 4) resulted in covalent attachment of PLP to the lysyl residue of the retinal oxidase and the PLP modified-retinal oxidase was cut with cyanogen bromide, and the polypeptides modified with PLP were further digested with trypsin. Two of the peptides modified with PLP were purified from the digested polypeptide mixture and their amino acid sequences were determined.

Chemical Modification of Pseudomonas ochraceae 4-Hydroxy-4-Methyl-2-0xoglutarate Aldolase by Diethyl Pyrocarbonate

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.

Chloroplast Intragenic Suppression Enhances the Low CO2/O2 Specificity of Mutant Ribulose-bisphosphate Carboxylase/Oxygenase

The competition between CO2 and O2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase limits net CO2 fixation in photosynthesis. In the green alga Chlamydomonas reinhardtii, a mutation in the chloroplast large-subunit gene reduces the CO2/O2 specificity of the enzyme by 37% and causes valine-331 to be replaced by alanine. Revertant selection identified an intragenic suppressor mutation that increases the CO2/O2 specificity of the mutant enzyme by 33%. This second-site mutation causes threonine-342 to be replaced by isoleucine. The complementing amino acid substitutions flank a catalytically essential lysyl residue at position 334. It thus appears that a number of amino acid residues can influence the CO2/O2 specificity of this bifunctional enzyme. The well defined chloroplast genetics of C. reinhardtii allows the interactions of these residues to be investigated.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Affinity labeling of the essential lysyl residue at the active site of enzymes by using nucleotide polyphosphate pyridoxal

We have discovered a new type of affinity labeling reagents for the nucleotide-binding site of protein by introducing an active site-directing moiety to pyridoxal 5′-phosphate. Uridine diphosphopyridoxal almost completely inactivated glycogen synthase in a time-dependent manner (K inact =25 µM;k 0=0.22 min−1). The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator Glc-6-P. The addition of cysteamine to the inactivated enzyme restored the original activity, whereas the treatment of the inactivated enzyme with NaBH4 resulted in the fixation of the label to the enzyme protein. A peptide containing the label was isolated after proteolysis, and sequenced as E-V-A-K*-V-G-G-I-(Y). Adenosine polyphosphopyridoxal considerably inactivated lactate dehydrogenase in a time-dependent manner. The degree of inactivation was dependent on the number of phosphate groups; 64% (N=2), 51% (N=3), and 35% (N=4) at a reagent concentration of 1 mM for 30 min. The inactivation was protected by NADH, but not by pyruvate. Although the inactivation was not completed, the reagent was stoichiometrically incorporated into enzyme protein concomitantly with the inactivation. Affinity chromatographic analysis of the inactivated enzyme of Blue-Toyopearl revealed the presence of several protein species. The ratio of those species changed according to the stage of inactivation.

Mitochondrial nicotinamide nucleotide transhydrogenase: Inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and l-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5′-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5′-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.

Chemical and kinetic characterization of tadpole back skin collagenase

The purified collagenase from tadpole ( Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNPProGlnGlyIleAlaGlyGln dArg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly-Ile bond and proved to be a poor substrate ( k cat K m , 1.21 h −1 μ m −1) when compared with native type I collagen in solution ( k cat K m , 40.6 h −1 μ m −1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145] . The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2-hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl 2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues.

Metabolism of glycerate-2,3-P 2—VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5′-phosphate produces the concurrent loss of the three activites of the enzyme: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. 3. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P 2. 4. 4. Substrate (glycerate-3-P) protects against pyridoxal-5′-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P 2) does not prevent inactivation. 5. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. 6. 6. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.

Chemical modification and ligand binding studies with Escherichia coli glutamate synthase.

The structure and function of Escherichia coli glutamate synthase were studied by ligand binding and chemical modification experiments. Binding of NADP+ was to a single dinucleotide site per alpha beta protomer with a Kd of approximately 5 microM. Phenylglyoxal modified an essential arginyl residue required for binding of NADP+. E. coli glutamate synthase thus employs a single dinucleotide binding site which functions in the NADPH to flavin electron transfer for glutamine-dependent glutamate synthase and for direct reduction of 2-iminoglutarate by NADPH in the NH3-dependent reaction. Binding of 2-oxoglutarate was complex. "Half of the sites" binding of 2-oxoglutarate (Kd less than 0.25 microM) was obtained in the absence of glutamine. Binding to half of the sites was pH independent. In the presence of glutamine, the 2-oxoglutarate binding ratio was approximately 1 equiv per protomer (Kd = 2-3 microM) at pH 7.5. This binding was pH dependent and varied between 0.43 equiv per protomer at pH 6.7 and 2.3 equiv per protomer at pH 9.0. Correlation of half of the sites binding with negative cooperativity for 2-oxoglutarate saturation indicates the utilization of low-Kd 2-oxoglutarate sites for NH3-dependent glutamate synthase. Binding of glutamine promotes a conformational change that exposes additional 2-oxoglutarate sites having a Kd of 2-3 microM which are utilized in the glutamine-dependent reaction. Chemical modification with pyridoxal 5'-phosphate caused inactivation of glutamine-dependent but not NH3-dependent glutamate synthase. Inactivation was ascribed to modification of one to two lysyl residues per protomer by Schiff base formation. The essential lysyl residue has a role in the binding of glutamine.

Chemical modification A probe of the structure and function of the subunits of DPN-dependent isocitrate dehydrogenase.

DPN-dependent isocitrate dehydrogenase is composed of three distinct types of subunits: alpha, beta, and gamma which have molecular weights of about 40,000 but differ in isoelectric points. The relationship of subunit diversity to function was probed by use of chemical modification. 3-Bromo-2-ketoglutarate, a substrate and affinity label for the active site of isocitrate dehydrogenase, was shown to cause significant modification of all types of subunits. The substrate affinity label, 3-ene-2-keto-glutarate, labels each of the subunits equally. Approximately equal labeling of subunits was also found upon modification by cyanate of an essential lysyl residue in the isocitrate binding site. When enzyme was inactivated by a carbodiimide in the presence of glycine ethyl ester, both glutamate and aspartate residues reacted, and labeling of each type of subunit occurred. These studies suggest that the structurally distinct subunits of DPN-dependent isocitrate dehydrogenase are functionally similar and each type of subunit contains a substrate binding site.

Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed in contrast to an earlier report [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. Subsequent to reduction with [3H]borohydride and tryptic digestion of the enzyme inactivated by PLP, the sole labeled peptide was purified by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. The peptide, recovered in good yield, appeared essentially homogeneous by amino acid analysis, peptide mapping, and sequencing. Automated Edman degradation established the peptide's sequence as Val-Leu-Gly-Arg-Pro-Glu-Val-Asp-Gly-Gly-Leu-Val-Val-Gly-Thr-Ile-Ile-(PLP)Lys -Pro-Lys instead of Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP)Lys-Gly-Thr-Leu-Val-Ile-Lys as reported by Robison et al. (1980) [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. The sequence -Ile-Lys-Pro-Lys- in the former is identical with that encompassing Lys-175 in the carboxylase/oxygenase from spinach, which reacts preferentially with PLP and two other affinity labels. This finding of homology greatly strengthens the supposition that Lys-175 in the spinach enzyme and the corresponding lysyl residue in the Rhodospirillum rubrum enzyme are active-site residues and furthermore increases the likelihood of their functionality in catalysis.

Chemical modification of coenzyme B 12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal- P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH 4. ϵ- N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH 4-reduced enzyme-pyridoxal- P complex. The relationship of inactivation vs pyridoxal- P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal- P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal- P results from irreversible dissociation of the enzyme into subunits upon pyridoxal- P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal- P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).

Identification of essential amino acid residues in [formula omitted] collagenase using chemical modification reactions

Clostridium histolyticum collagenase has been chemically modified with a series of reagents to identify essential amino acid residues. The activity of the enzyme is not significantly altered by the seryl reagents diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, the cysteinyl reagents p-chloromercuribenzoate and iodoacetamide, or the arginyl reagents butanedione and phenylglyoxal. The enzyme is inactivated by 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3′-sulfonate, indicating the presence of an essential carboxyl residue. Both acetylimidazole and tetranitromethane inactivate the enzyme and the acetylimidazole reaction is reversed by hydroxylamine, indicating that collagenase contains an essential tyrosyl residue. In addition, acylation of the enzyme by diethylpyrocarbonate, diketene and acetic anhydride markedly lowers activity, which cannot be restored by hydroxylamine. This indicates that collagenase contains an essential lysyl residue, a conclusion supported by the fact that trinitrobenzene sulfonate also inactivates the enzyme.

Phenol hydroxylase from yeast: a lysyl residue essential for binding of reduced nicotinamide adenine dinucleotide phosphate.

The inducible enzyme phenol hydroxylase from Trichosporon cutaneum is a FAD-containing monooxygenase which catalyzes the NADPH-dependent hydroxylation of phenol to catechol. The enzyme contains 16 cysteinyl residues, 6--8 of which are essential for retention of FAD and for activity. The complete amino acid composition is now reported as well as the results of studies with amino group reagents. A number of amino group reagents inhibit the enzyme severely, most of them with a concomitant, more or less extensive release of FAD. P-pyridoxal inhibits the enzyme specifically, without affecting its FAD content. The P-pyridoxal modified enzyme has a characteristic absorption peak at 325 nm indicating the presence of a N epsilon-pyridoxyllysyl derivative. Such a derivative was identified in hydrolysates of the modified enzyme by means of column chromatography. The results obtained with P-pyridoxal-modified enzyme indicate that a lysyl residue is essential for activity by being involved in binding of the co-substrate NADPH. These results are corroborated by kinetic studies showing competition between P-pyridoxal and NADPH for the binding site. The reactivity of the essential lysyl residue toward P-pyridoxal is significantly increased in the presence of phenol. Inhibition by excess phenol shifts toward lower concentrations in the presence of P-pyridoxal. On the basis of the present results together with previous findings, we propose that phenol acts as a substrate effector by causing a conformation change which exposes a reactive lysyl residue with a concomitant burying of the essential SH groups and a tighter attachment of FAD.

Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate

We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).

Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase

Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho- d-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco ( Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO 2 and Mg 2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg 2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO 2 and Mg 2+. Incubation of the carboxylase with potassium [ 14C]cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioative carboxylase indicated that the reagent carbamylates both NH 2-terminal groups and lysyl residues in the large and small subunits. Comparison ot the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO 2 binding sites on the enzyme.

Sequences of two active-site peptides from spinach ribulosebisphosphate carboxylase/oxygenase

Two tryptic peptides from spinach ribulosebisphosphate carboxylase/oxygenase that contain the essential lysyl residues derivatized by the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate were subjected to sequence analyses. The sequences of these peptides are -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys-Thr-Ile-Lys-Pro-Lys- and -Leu-Ser-Gly-Gly-Asp-His-Ile-His-Ser-Gly-Thr-Val-Val-Gly-Lys-Leu-Glu-Gly-Glu-Arg-, respectively. The reagent moiety is covalently attached to the internal lysyl residue in each peptide.