Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K ( WRK ). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK . SBP was a strong protector, displaying a dissociation constant (Kd) of 3 microM with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 +/- 700 M-1 cm-1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.     

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.     

Inactivation of ribulosebisphosphate carboxylase/oxygenase from rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both $\text{Rhodospirillum}\text{,}\text{rubrum}$ and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with [³H]NaBH₄, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the $\text{Rhodospirillum}\text{,}\text{rubrum}$ enzyme and less than one residue per protomeric unit of the spinach enzyme.     

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.     

Comparative affinities of the epimeric reaction-intermediate analogs 2- and 4-carboxy-D-arabinitol 1,5-bisphosphate for spinach ribulose 1,5-bisphosphate carboxylase

2-Carboxy-3-keto-D-arabinitol 1,5-bisphosphate is a tightly bound intermediate of the carboxylase reaction of ribulosebisphosphate carboxylase/oxygenase. Two stereoisomers of an analog of this intermediate, 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP) and 4-carboxy-D-arabinitol 1,5-bisphosphate (4CABP), are exceptionally potent, virtually irreversible inhibitors of the spinach carboxylase, presumably due to their structural similarity to the gem-diol (hydrated carbonyl at C-3) form of the intermediate. Incubation of the enzyme with either leads to time-dependent loss of activity. Inhibition of the enzyme is biphasic, with initial dissociation constants of 0.47 and 0.19 microM and maximal rates for tight complex formation of 2.2 and 1.8 min-1 for 2CABP and 4CABP, respectively. These values give second-order rate constants for tight complex formation of 7.8 x 10(4) and 1.6 x 10(5) M-1 s-1. To determine the overall affinity of the spinach enzyme for 2CABP and 4CABP, the release rates were determined by dual isotope exchange (3H-inhibitor complex with free 14C-inhibitor). Exchange half-times of 1.82 and 530 days were observed for 4CABP and 2CABP, respectively. Overall dissociation constants of 28 pM (2.8 x 10(-11) M) and 190 fM (1.9 x 10(-13) M) were calculated from these dissociation rates together with the rates of association determined by inactivation kinetics. The difference in affinity of 2CABP and 4CABP corresponds to 2.9 kcal/mol, presumably reflecting the difference in interaction of the enzyme with the two hydroxyls of the intermediate's gem-diol. The kinetic behavior of these two inhibitors, in particular the rather slow maximal rates of association, are consistent with the expected behavior of analogs of a labile intermediate of an enzymic reaction that is far more stable than a transition state.     

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO2/Mg2+-activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 mM and a maximum pseudo-first-order rate constant of 7 X 10(-3) s-1. The rate constant for reactivation was 1.2 X 10(-2) s-1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme X CO2 X Mg2+ X CABP.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

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)     

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.     

Reversible desensitization of phosphoenolpyruvate carboxylase to multiple effectors by butanedione.

Chemical modification of phosphoenolpyruvate carboxylase [EC 4.1.1.31] of Escherichia coli W with 2,3-butanedione, an arginyl residue reagent, results in an inactivation of the enzyme. The inactivation proceeds following pseudo-first order kinetics. DL-Phospholactate, a substrate analog, effectively protects the enzyme from the inactivation. The enzyme modified in the presence of DL-phospholactate or in its absence is completely desensitized to fructose 1,6-bisphosphate and GTP, allosteric activators for the enzyme. At the same time, the sensitivities to acetyl coenzyme a, laurate and L-aspartate are considerably decreased. Resensitization is attained, however, upon removal of excess butanedione and borate by gel filtration, concomitant with the restoration of the catalytic activity.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues.

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.     

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.     

Further studies on the subunit structure of Chromatium ribulose-1,5-phosphate carboxylase.

Upon alkali exposure Chromatium ribulose-1,5-bisphosphate carboxylase dissociates into constituent subunits, a catalytic oligomer of the larger subunit, A8, and monomeric form of the small subunit B. By sedimentation equilibrium molecular weights of the native enzyme and the catalytic oligomer produced by an alkali treatment were estimated to be 5.11 x 10 5 and 4.29 x 10 5, respectively. To provide information on reversibility of the dissociation by determining whether the enzymically inactive small subunit B of the whole enzyme molecule did indeed exchange with exogenously added subunit B a radioisotopic method was used. After initial alkaline dialysis at pH 9.2 of a mixture of a nonlabeled native enzyme preparation and 14C-labeled subunit B, and the subsequent dialysis at pH 7.0, incorporation of 14C into the recovered native enzyme was determined. Without the alkaline treatment there was no detectable exchange, while after alkaline dialysis for 5 and 10 hr the subunit B exchange was 89 and 82%, respectively. Rabbit antiserum prepared against the catalytic oligomer of the spinach ribulose-1,5-bisphosphate carboxylase, anti-(A) (spinach), inhibited the Chromatium carboxylase and oxygenase activities. This result together with the identical immunoprecipitation lines on an agar plate formed between the antiserum and the Chromatium carboxylase and between the antiserum and the catalytic subunit of the Chromatium enzyme strongly indicated structural near identity of the catalytic subunits of the spinach and Chromatium carboxylase molecules. Results also show that the catalytic site of the Chromatium ribulose-1,5-bisphosphate carboxylase and oxygenase exists in the large polypeptide chain.     

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.     

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.     

Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase

Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.     

Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase

Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.