Cryoenzymology of human plasmin catalysis: comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates.
A comparative study of nucleophilic (methanol), aprotic (dimethyl sulfoxide), and protic but non nucleophilic (ethylene glycol, ethylene glycol/dimethylformamide) solvents on the catalytic and structural properties of human plasmin has been made. All four solvent systems are potentially suitable as cryosolvents for plasmin catalysis at subzero temperatures although the solubility of plasmin is limited in the methanol and dimethyl sulfoxide systems. Each cryosolvent system caused minor effects on the catalytic properties of the enzyme, which could be rationalized in terms of the known physical properties of the cosolvent. Solvent systems containing ethylene glycol induce a minor conformational change which increases the catalytic efficiency of plasmin. The cosolvent effects on Km and Ki indicate that electrostatic interactions dominate the binding of both substrates and inhibitors such as benzamidine. A change in slope of the Arrhenius plots for catalysis, reflecting a temperature-induced isomerization, is observed around 0 degree C; the energies of activation being 13 +/- 2 kcal mol-1 at higher temperatures and 19 +/- 2 kcal mol-1 at subzero temperatures, and essentially independent of solvent. Deacylation was shown to be the rate-limiting step in the hydrolysis of specific p-nitrophenyl ester substrates. Previous stopped-flow studies at room temperature provided observations suggesting that a tetrahedral intermediate could be detected in the plasmin-catalyzed hydrolysis of p-nitroanilide substrates. Experiments at subzero temperatures with such substrates failed to reveal any buildup of a tetrahedral intermediate under the experimental conditions.
D000813:Anilides; D002384:Catalysis; D004121:Dimethyl Sulfoxide; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000378:metabolism*; D005615:Freezing; D006801:Humans; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D008970:Molecular Weight; D009596:Nitrophenols; D010958:Plasminogen / Q000032:analysis / Q000302:isolation & purification; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000813:Anilides; D009596:Nitrophenols; D012997:Solvents; D010958:Plasminogen; D005341:Fibrinolysin; D000432:Methanol; D004121:Dimethyl Sulfoxide