scholarly journals Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit

1990 ◽  
Vol 267 (2) ◽  
pp. 531-533 ◽  
Author(s):  
U Tisljar ◽  
A J Barrett
Keyword(s):  
Catalytic Activity ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Rat Testis ◽  
Free Thiol

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

scholarly journals Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

2005 ◽  
Vol 386 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sandra MÜLLER ◽  
Manuela SCHÖTTLER ◽  
Sylvia SCHÖN ◽  
Christian PRANTE ◽  
Thomas BRINKMANN ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

THE PYRUVIC PHOSPHOFERASE OF ERYTHROCYTES: I. PROPERTIES OF THE ENZYME AND ITS ACTIVITY IN ERYTHROCYTES OF VARIOUS SPECIES

1955 ◽  
Vol 33 (1) ◽  
pp. 38-45 ◽  
Author(s):  
P. F. Solvonuk ◽  
H. R. Collier
Keyword(s):  
Mammalian Species ◽  
Thiol Groups ◽  
Newborn Rats ◽  
Free Thiol ◽  
The Mean ◽  
Very High

Mammalian erythrocytes contain a pyruvic phosphoferase (PPFase) which is activated by K+ and Mg++ and inhibited by Na+ and Ca++. The K+ can be replaced by Rb+ or NH4+, and the Mg++ can be partially replaced by Mn++ or Co++ as activators of the enzyme. The PPFase apparently requires free thiol groups for its activity, as it is completely inhibited by 10−4 M p-chloromercuribenzoate and this inhibition is partially reversed by glutathione. The mean PPFase of the erythrocytes of six mammalian species was determined and found to be in the following order of decreasing activity: man, rat, dog, rabbit, cat, ox. The erythrocytes of chicks and of newborn rats showed a very high PPFase activity.

scholarly journals Purification and properties of two glyoxylate reductases from a species of Pseudomonas

1966 ◽  
Vol 101 (3) ◽  
pp. 781-791 ◽  
Author(s):  
LN Cartwright ◽  
RP Hullin
Keyword(s):  
Equilibrium Constants ◽  
Maximal Activity ◽  
High Order ◽  
Thiol Groups ◽  
Molecular Weights ◽  
Free Thiol ◽  
Purification And Properties ◽  
Keto Acids ◽  
Michaelis Constants

1. Two enzymes that catalyse the reduction of glyoxylate to glycollate have been separated and purified from a species of Pseudomonas. Their molecular weights were estimated as 180000. 2. Reduced nicotinamide nucleotides act as the hydrogen donators for the enzymes. The NADH-linked enzyme is entirely specific for its coenzyme but the NADPH-linked reductase shows some affinity towards NADH. 3. Both enzymes convert hydroxypyruvate into glycerate. 4. The glyoxylate reductases show maximal activity at pH6.0-6.8, are inhibited by keto acids and are strongly dependent on free thiol groups for activity. 5. The Michaelis constants for glyoxylate and hydroxypyruvate were found to be of a high order. 6. The reversibility of the reaction has been demonstrated for both glyoxylate reductases and the equilibrium constants were determined. 7. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.

scholarly journals The reaction of aldolase with 2-methylmaleic anhydride

1970 ◽  
Vol 116 (5) ◽  
pp. 843-849 ◽  
Author(s):  
I. Gibbons ◽  
R. N. Perham
Keyword(s):  
Enzyme Activity ◽  
Maleic Anhydride ◽  
Enzymic Activity ◽  
Side Reactions ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Amino Groups ◽  
Partial Loss ◽  
Reaction Proceeds ◽  
Citraconic Anhydride

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.

scholarly journals Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine

1983 ◽  
Vol 215 (3) ◽  
pp. 513-518 ◽  
Author(s):  
A R S Prasad ◽  
J Ybarra ◽  
J S Nishimura
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Adenine Nucleotide ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Thiol Groups ◽  
Binding Region ◽  
Nucleotide Analogue ◽  
Disulphide Bond Formation ◽  
Succinyl Coa Synthetase

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). Optimal inactivation by 5′-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5′-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5′-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5′-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5′-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5′-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5′-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.

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