Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase

2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.     

Kinetics of the reaction between 5,5′-dithiobis[2-nitrobenzoic acid] and the sulphydryl group in Zn-dependent β-lactamase II

The kinetics of the reaction of the thiol residue in Zn²⁺-dependent β-lactamase II with 5,5′-dithiobis[2-nitrobenzoic acid], and the concomitant inactivation revealed that both events take place at the same rate. The inactivation could not be reverted by incubation with Zn²⁺ or by using a substrate concentration about eight times the K_m of the enzyme. EDTA incubation also produced inactivation of the enzyme, although it was reverted by increasing the substrate concentration in the assay. A dual role is proposed for Zn²⁺ in β-lactamase. The kinetic analysis of the thiol modification and the concomitant inactivation is in agreement with previous reports on the implication of the metal ion in catalysis. A role in stabilizing the native structure of the enzyme is also suggested.     

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.     

Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl 2-tetradecylglycidate (methyl palmoxirate) and its characterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria

Methyl-2-tetradecylglycidic acid (MeTDGA) has been hypothesized to inhibit fatty acid oxidation by irreversible, active site-directed inactivation of carnitine palmitoyltransferase A after being converted to TDGA-CoA. Using synthetic TDGA-CoA, this hypothesis has been confirmed. Assessing enzyme inhibition in an isolated rat liver mitochondrial system, TDGA-CoA (synthetic or enzyme prepared) was more potent than TDGA or MeTDGA and retained activity in the absence of CoA or Mg2+-ATP. It inhibited palmitoyl-CoA but not palmitoyl carnitine oxidation. Enzyme inactivation was exponential, stereospecific, and fast (t0.5 = 38.5 s with 100 nM (R)-TDGA-CoA). TDGA-CoA was identified as a complexing type irreversible inhibitor (Ki approximately 0.27 microM) by the double reciprocal relationship between the pseudo-first order inactivation rate and its concentration, by the inverse dependence of the second order rate constant on its concentration, and by the independence of the first order rate from the enzyme concentration. Palmitoyl-CoA, CoA, and malonyl-CoA protected the enzyme, while L-carnitine and palmitoyl-L-carnitine were without effect. [3-14C] TDGA-CoA labeled a protein, Mr = 90,000, with a time course which paralleled that of enzyme inhibition; maximum specific binding was 16 pmol/mg of mitochondrial protein.     

Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate

Maize malic enzyme was rapidly inactivated by micromolar concentrations of cupric nitrate in the presence of ascorbate at pH, 5.0. Ascorbate or Cu2+ alone had no effect on enzyme activity. The substrate L-malate or NADP individually provided almost total protection against Cu2+-ascorbate inactivation. The loss of enzyme activity was accompanied by cleavage of the enzyme. The cleaved peptides showed molecular mass of 55 kDa, 48 kDa, 38 kDa, and 14 kDa. Addition of EDTA, histidine and imidazole provided protection. The results of protection experiments with sodium azide, DABCO and catalase suggested that reactive oxygen species were generated resulting in loss of enzyme activity. This was further supported by experiments showing that the rate of enzyme inactivation was higher in D2O than in water. It is suggested that maize malic enzyme is modified by reactive oxygen species like singlet oxygen and H2O2 generated by Cu2+-ascorbate system and the modified amino acid residue(s) may be located at or near the substrate-binding site of the enzyme.     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system.

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).     

2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors

Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake. This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate. This is achieved by use of 2-deoxy-2-fluoro-D-glycosyl fluorides, for which the rate of hydrolysis of the fluoroglycosyl enzyme intermediate is extremely slow, resulting in accumulation of the intermediate. Eleven different glycosidases were tested with their corresponding 2-deoxy-2-fluoro-D-glycosyl fluorides. Eight of the eleven were inactivated, four of them according to pseudo first-order kinetics and four according to a more complex kinetic scheme. The specificity of these inhibitors was investigated by assaying for inhibition of one enzyme with four different 2-deoxy-2-fluoro-D-glycosyl fluorides. Large differences in inactivation rate were observed which paralleled previously observed substrate specificities.     

Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.     

Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.     

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.     

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.     

The effect of 2H2O on the quaternary structure of the fatty acid synthetase multienzyme complex

The effect of 2H2O on the cold-induced dissociation and cold-induced inactivation of fatty acid synthetase from lactating mammary gland has been studied. The rate constants for these reactions were almost identical and were lowered in media containing 2H2O. The results have been interpreted to indicate that inactivation of the enzyme in the cold is a direct result of dissociation into subunits and that maintenance of the native structure of the enzyme is limited by the strength of hydrophobic bonds.     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. EIIFru possesses a Zn2+-binding site and a dithiol/disulfide redox centre

Two interrelated sites have been detected on the fructose carrier in Rhodopseudomonas sphaeroides: an activity-linked dithiol and a Zn2+-binding site. Binding of Zn2+ brings EIIFru into a new conformation that to some extent mimics the conformation of phosphorylated EIIFru, an essential intermediate in the turnover of the enzyme. Binding of zinc to EIIFru or phosphorylating the enzyme protects it against trypsin inactivation relative to the dephosphorylated zinc-free enzyme. A dithiol is essential for activity. Interchanges between the redox states of the enzyme can be brought about by dithiothreitol and ferricyanide, but not, or very slowly, by molecular oxygen. The dithiol is protected, in the EIIFru-Zn2+ complex, against alkylation by MalNEt, reversible oxidation by Fe(CN)6(3-) and Cu2+, irreversible oxidation by Cu2+. The pK value of the activity linked thiol is 7.8. Protection experiments show that the dithiol is not located in any of the substrate-binding sites. The redox state of the enzyme does not influence the rate of inactivation of EIIFru by trypsin.     

Importance of SH groups in catalysis by bovine brain acid phosphatase.

The rate of inactivation of acid phosphatase (EC 3.1.3.2) from bovine brain by dithiobis-(2-nitrobenzoic acid) (Nbs2) is identical to the rate of titration of one of the two SH groups of this enzyme. The rate of inactivation of the enzyme by Nbs2 is pH dependent and, at 300 mM NaCl, can be described by the reaction of a single SH group of pK 8.4. At low ionic strength the pK determined from the k inactivation vs. pH profile is 7.7 and the results deviate markedly from the predicted values at pH values less than or equal to 6. The decrease of V upon addition of salts is paralleled by the decrease of inactivation rate by Nbs2. The relevance of SH groups in catalysis by bovine brain acid phosphatase is discussed in terms of these data.