ABSTRACT
The three cysteine residues per subunit of pig muscle phosphoglucose isomerase show different reactivities toward various sulfhydryl reagents. The organomercurial, p-mercuribenzoate, can titrate two of the sulfhydryl groups under nondenaturing conditions. 2,2'-Dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, methyl 2-pyridyl disulfide, and 2-(2'-pyridylmercapto)mercuri-4-nitrophenol all label only one sulfhydryl group under the same conditions, whereas iodoacetic acid does not react with any of the sulfhydryl groups except when the enzyme is fully denatured. It is concluded, therefore, that charge, rather than steric restraint, is the determining factor for the differences seen in the modification patterns of the enzyme by these reagents. When enzyme was first labeled with 2,2'-dithiodipyridine and subsequently with p-mercuribenzoate, it was found that the latter, in a secondary process, will stoichiometrically react with the anion released by the former after the initial reaction with cysteine. The differences in reactivity of the cysteine residues toward the referred-to reagents have been exploited to specifically modify each of the three individual cysteine residues of pig muscle phosphoglucose isomerase.
Subject(s)
Cysteine/analysis , Glucose-6-Phosphate Isomerase/metabolism , Muscles/enzymology , Sulfhydryl Reagents/pharmacology , Amino Acid Sequence , Animals , Hydrogen-Ion Concentration , In Vitro Techniques , Protein Denaturation , SwineABSTRACT
Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.
Subject(s)
Carbonic Anhydrases/isolation & purification , Muscles/enzymology , Amino Acids/analysis , Animals , Kinetics , Molecular Weight , Spectrophotometry, Ultraviolet , SwineABSTRACT
Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.
Subject(s)
Arginine/metabolism , Carbonic Anhydrases/metabolism , Muscles/enzymology , Phosphoric Monoester Hydrolases , 4-Nitrophenylphosphatase/metabolism , Animals , Binding Sites , Carbon Dioxide/metabolism , Esterases/metabolism , Kinetics , Phenylglyoxal/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Binding , SwineABSTRACT
The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO2 hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO2 hydration was found to be 6000 sec-1 with a Km of 83 mM for CO2. Cyanate inhibition (Ki, 3 microM) and acetazolamide inhibition (Ki, 44 microM) were both found to be noncompetitive with respect to CO2. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large Ki for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinate zinc in the catalytic mechanism of CO2 hydration for CA III.
Subject(s)
Acetazolamide/pharmacology , Carbonic Anhydrase Inhibitors , Cyanates/pharmacology , Muscles/enzymology , Animals , Carbon Dioxide/metabolism , Erythrocytes/enzymology , Kinetics , SwineSubject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Isoenzymes/antagonists & inhibitors , Muscles/enzymology , Amino Acids/analysis , Animals , Carbonic Anhydrases/isolation & purification , Cattle , Cyanates/pharmacology , Isoenzymes/isolation & purification , Kinetics , Phenylglyoxal/metabolism , Phenylglyoxal/pharmacology , Protein Binding , Rabbits , Species Specificity , SwineABSTRACT
Rabbit muscle phosphoglucose isomerase was modified with phenylglyoxal or 2,3-butanedione, the reaction with either reagent resulting in loss of enzymatic activity in a biphasic mode. At slightly alkaline pH butanedione was found to be approximately six times as effective as phenylglyoxal. The inactivation process could not be significantly reversed by removal of the modifier. Competitive inhibitors of the enzyme protected partially against loss of enzyme activity by either modification. The only kind of amino acid residue affected was arginine. However, more than one arginine residue per enzyme subunit was found to be susceptible to modification by the dicarbonyl reagents. From protection experiments it was concluded (i) that both modifiers react specifically with an arginine in the phosphoglucose isomerase active site and nonspecifically with one or more arginine residues elsewhere in the enzyme molecule, (ii) that modification at either loci causes loss of catalytic activity, and (iii) that butanedione has a higher preference for active site arginine than for arginine residues outside of the catalytic center whereas the opposite is true for phenylglyoxal.
Subject(s)
Aldehydes/pharmacology , Arginine/metabolism , Epoxy Compounds/pharmacology , Ethers, Cyclic/pharmacology , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Muscles/enzymology , Phenylglyoxal/pharmacology , Animals , Binding Sites/drug effects , Catalysis , Glucose-6-Phosphate Isomerase/metabolism , Hydrogen-Ion Concentration , Kinetics , RabbitsABSTRACT
Pig muscle phosphoglucose isomerase modified with pyridoxal 5'-phosphate under conditions that cause at least 90% inactivation of its catalytic activity was found to incorporate about 1.5 eq of pyridoxal 5'-phosphate per subunit. After digestion with thermolysin, two pyridoxal 5'-phosphate-containing peptides were isolated and their amino acid sequences were determined to be Leu-Gly-pyridoxyl-Lys-Gln and Ile-Ala-Ser-pyridoxyl-Lys-Thr.
Subject(s)
Muscles/enzymology , Phosphoglucomutase/isolation & purification , Pyridoxal Phosphate/analysis , Amino Acids/analysis , Animals , Peptide Fragments/analysis , Protein Binding , Swine , ThermolysinSubject(s)
4-Nitrophenylphosphatase/metabolism , Carbonic Anhydrases/metabolism , Isoenzymes/metabolism , Muscles/enzymology , Phosphoric Monoester Hydrolases/metabolism , Animals , Carbonic Anhydrase Inhibitors , Drug Stability , Hydrogen-Ion Concentration , Protein Denaturation , Rabbits , Substrate SpecificityABSTRACT
Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.
Subject(s)
Glucose-6-Phosphate Isomerase/metabolism , Muscles/enzymology , Amino Acid Sequence , Animals , Binding Sites , Chemical Phenomena , Chemistry , Macromolecular Substances , Models, Biological , Models, Molecular , Molecular Weight , Peptide Fragments , Species Specificity , X-Ray DiffractionABSTRACT
A new covalent active site reagent, 1-chloro-2-oxo-6-hexanol 6-phosphate, has been synthesized from glutaric acid monomethyl ester and characterized by NMR spectroscopy. Inactivation of phosphoglucose isomerase, when incubated with various modifier concentrations, was found to be pseudo first order with respect to enzyme concentration (half-life of inactivation 6 h at pH 7.5 (30 degrees C) and 2.0 muM active site concentration) but showed saturation kinetics for the dependence on inactivator concentration. This saturation phenomenon demonstrates the occurrence of a reversible enzyme-inhibitor complex (Kdiss = 14.2 mM) preceding the irreversible inactivation via the chloromethyl oxo groups. Substrate or competitive inhibitors such as 6-phosphogluconate or 5-phosphoarabinonate protect against inactivation of the isomerase by the modifying reagent.
Subject(s)
Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Organophosphates/chemical synthesis , Organophosphorus Compounds/chemical synthesis , Animals , Binding Sites , Indicators and Reagents , Kinetics , Magnetic Resonance Spectroscopy , Methods , Muscles/enzymology , Organophosphates/pharmacology , Protein Binding , RabbitsABSTRACT
A large-scale purification procedure for phosphoglucose isomerase from pig skeletal muscle is described. It consists of two fractionations by selective precipitation and two ion exchange chromatography steps yielding an end product of approximately 900 units (micromoles of substrate converted to product per min per mg of protein, at 30 degrees) specific activity. The method separates three isoenzymic forms with an overall recovery of about 30% of the original total enzyme activity in the form of Isoenzyme III, the latter being the predominant enzyme species.
Subject(s)
Glucose-6-Phosphate Isomerase/isolation & purification , Isoenzymes/isolation & purification , Muscles/enzymology , Animals , Carbonic Anhydrases/isolation & purification , Chromatography, Ion Exchange/methods , Crystallization , Fractional Precipitation/methods , SwineSubject(s)
Carbonic Anhydrases , Muscles/enzymology , Animals , Cattle , Circular Dichroism , Erythrocytes/enzymology , Humans , Isoenzymes , Protein Conformation , Rabbits , Species SpecificityABSTRACT
Rabbit muscle carbonic anhydrase III, a recently discovered third isoenzyme (possibly muscle specific) of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) (Register, A.M., Koester, M.K. and Noltmann, E.A. (1978) J. Biol. Chem. 253, 4143--4152) has been subjected to isoelectric focusing. When monomer samples, shown to be homogeneous by both ion-exchange and molecular sieve chromatography, were analyzed by this technique, three subspecies were produced, which were similar in amino acid composition and specific CO2 hydratase activity. In addition to having either monomer or dimer status, the subspecies differed in the extent of oxidation of their sulhydryl groups and in their isoelectric pH values (9.3, 8.8, and 8.4, respectively). Also, the presence of dithiothreitol will affect their relative concentrations. These subforms are therefore designated as pseudoisoenzymes and are considered to be neither genetically nor functionally separate enzyme species.
Subject(s)
Carbonic Anhydrases/metabolism , Isoenzymes/metabolism , Muscles/enzymology , Amino Acids/analysis , Animals , Carbonic Anhydrases/isolation & purification , Dithiothreitol , Isoenzymes/isolation & purification , Kinetics , RabbitsABSTRACT
A stable analogue has been prepared of the enediolate anion believed to occur transiently in the reaction of phosphoglucose isomerase. This compound, 5-phosphoarabinonate, is the strongest known competitive inhibitor of the enzyme (Ki = 3 times 10(-7) M below pH 7). A distinctive pH dependence of binding, also found for two other aldonic acid omega-phosphates, 6-phosphogluconate and 4-phosphoerythronate, involves pertubation of a pKa from 7.0 in the free enzyme to 9.0 in the enzyme-inhibitor complex. This perturbation may reflect a catalytically advantageous increase in basicity which occurs around the transition state of the normal enzymatic reaction.