Reaction of ribulosebisphosphate carboxylase from rhodospirlilum rubrum with the potential affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-biphosphate.

Under mild conditions, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate rapidly and irreversibly inactivates ribulosebisphosphate carboxylase from $\text{Rhodospirillum rubrum}$. The substrate ribulosebisphosphate protects the enzyme against inactivation. Incorporation of reagent has been quantitated by reduction of the modified carboxylase with [³H]NaBH₄. Based on the difference in the levels of incorporation found in the inactivated enzyme as compared with the protected enzyme, loss of enzymic activity results from the modification of about 0.4 residue per catalytic subunit. Analyses of hydrolysates demonstrate that both cysteinyl and lysyl derivatives are present in the inactivated carboxylase; the protected sample contains about the same amount of modified cysteine but little of the modified lysine. Thus, inactivation appears to correlate with derivatization of lysyl residues.     

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 CO2 and Mg2+ 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 Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. 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 radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of 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 CO2 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.     

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).     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.     

Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase

Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.     

Essential arginyl residues in yeast phosphoglyceromutase.

Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.     

Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

[3H] Diethyl pyrocarbonate was synthesized [Melchior, W. B., & Fahrney, D. (1970) Biochemistry 9, 251-258] from [3H] ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM Mg2+, and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with our earlier experiments [Saluja, A. K., & McFadden, B. A. (1982) Biochemistry 21, 89-95], suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.     

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate 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.     

Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.     

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.     

Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.     

Reaction of phenylglyoxal and (p-hydroxyphenyl) glyoxal with arginines and cysteines in the alpha subunit of tryptophan synthase

The alpha subunit of tryptophan synthase from Escherichia coli is inactivated by phenylglyoxal and by (p-hydroxyphenyl)glyoxal. The use of these chemical modification reagents to determine the role of arginyl residues in the alpha subunit of tryptophan synthase has been complicated by our finding that these reagents react with sulfhydryl groups of the alpha subunit, as well as with arginyl residues. Analyses of the data for incorporation of phenyl[2-14C]glyoxal, for inactivation, and for sulfhydryl modification in the presence and absence of indole-3-glycerol phosphate indicate that two sulfhydryl groups and one arginine are essential for the activity. Our finding that the substrate protects the single essential arginyl residue but not the two sulfhydryl groups is consistent with the observed kinetics of partial protection by substrate or by a substrate analogue, indole-3-propanol phosphate. In contrast to phenylglyoxal, (p-hydroxyphenyl)glyoxal modifies two to three sulhydryl groups that are not protected by indole-3-glycerol phosphate and modifies none of the arginyl residues that are modified by phenylglyoxal.     

Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.     

Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.     

Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study

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.     

Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues.

Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.     

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.