No abstract

No abstract

Superoxide dismutase, which catalyzes the reaction Or + Or + 2H+ -H202 + 02, at rates which approach the diffusion limit, appears to be an important component of the defenses which have evolved to deal with the toxicity of oxygen. This enzyme contains Cu2+ and Zn2+. The apoenzyme is inactive and Cu2+ can restore activity. Analysis of the superhyperfine lines of the epr (Rotilio et al. (1971), Biochemistry 10, 616) spectrum suggests that the ligand field of the Cu2+, in this enzyme, contains three nitrogenous groups. Chemical modifications of specific residues have been applied in an attempt to identify these nitrogenous ligands. The apoenzyme was inactivated by exposure to light in the presence of a photosensitizing dye. This photoinactivation was paralleled by a decrease in histidine content, whereas other amino acid residues, including cysteine and methionine, remained unchanged. The holoenzyme was completely resistant to photosensitized inactivation and to photosensitized oxidation of histidine residues, under otherwise identical conditions. The apoenzyme was protected by stoichiometric amounts of either T A he chemical functionalities which are essential for the activity of enzymes have frequently been identified through the application of reagents which preferentially attack specific chemical groupings (Cohen, 1968; Glazer, 1970). In such studies, a loss of activity which correlates with modification of a residue has usually been taken as evidence of the importance of that residue for the catalytic function of the enzyme under study. If the reactive residue is protected and the inactivation prevented by substrate, substrate analog or prosthetic group then the residue may be part of the active site of the enzyme.Histidine and tryptophan have been found to be quite susceptible to photosensitized oxidation, whereas tyrosine, cysteine, and methionine are much less reactive and the other amino acid residues found in proteins are resistant (Weil and Buchert, 1951;Weil et al., 1952Weil et al., , 1953. Martinez-Carrion et al. (1967) have used photosensitized oxidation to establish the importance of specific histidine residues for the catalytic action of aspartic aminotransferase, and in this case histidine was the only residue significantly attacked.Diazotized sulfanilamide or sulfanilic acid couples with the imidazole ring of histidine to give colored products whose absorbance can be used to quantitate histidine in solutions (Macpherson, 1942) or to localize histidine-containing peptides on paper chromatograms (Baldridge and Lewis, 1953). A similar reagent, diazonium-1 /f-tetrazole, has been used to derivatize histidine residues in proteins (Horinishi et al., 1964)

No abstract

The three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit blood coagulation with different potencies. The strongly anticoagulant basic isoenzyme CM-IV inhibits the prothrombinase complex, whereas the weakly anticoagulant isoenzymes CM-I and CM-II do not. To determine the role of enzymatic activity of the phospholipases in the inhibition of prothrombinase, we varied the time of incubation of each of these isoenzymes with the prothrombinase complex. The inhibition by CM-IV did not increase with time of incubation. CM-I and CM-II failed to inhibit the complex, even with complete hydrolysis of phospholipids in the assay mixture. After alkylation of its active-site histidine, CM-IV lost 97% of its enzymatic activity but retained 60% of its inhibitory potency on prothrombinase. CM-IV also inhibited prothrombinase activity in the absence of phospholipids, whereas CM-I and CM-II did not. The inhibition of the prothrombinase complex by CM-IV is thus not due to its binding to or hydrolysis of phospholipids. The kinetics of CM-IV inhibition of the prothrombinase complex in both the presence and absence of phospholipids was noncompetitive. This inhibition can be explained by binding of CM-IV to either factor Va or Xa, or both, to inhibit the complex. CM-IV differs from previously described nonenzymatic anticoagulants that are proteinase inhibitors or that inhibit the coagulation complexes by interfering with the binding of clotting factors to phospholipids. We conclude that the basic enzyme, CM-IV, inhibits the prothrombinase complex by a novel mechanism independent of enzymatic activity.