Effect of cryosolvents and subzero temperatures on the hydrolysis of L-leucine-p-nitroanilide by porcine kidney leucine aminopeptidase.
The hydrolysis of L-leucine-p-nitroanilide by porcine kidney leucine aminopeptidase in aqueous mixed-solvent systems containing methanol, ethanol, dimethyl sulfoxide, and dimethylformamide has been investigated in the -30 to -23 degrees C temperature range. At 23 degrees C and pH* values in the 8-10 range, the enzyme is stable for over 25 h in solutions containing 50% v/v of any of these four cosolvents. Measurements of the tryptophan fluorescence of the enzyme at pH* 9.0 confirm that the enzyme is not denatured under these conditions. KM increases exponentially and kcat decreases linearly with increasing cosolvent concentration. Methanol, in particular, has a very small effect on KM. Ultrafiltration experiments demonstrate that there is no dissociation of monomers of the enzyme brought about by the presence of 50% v/v methanol or dimethyl sulfoxide. Preliminary tests with the partition method provide no evidence for an acyl-enzyme intermediate. The effect of pH* on kcat and KM in 50% v/v methanol is very similar to the effect of pH on these kinetic constants in aqueous solution. Lowering the temperature from 23 to 0 degree C does not alter the shape of the pH* profile obtained in 50% v/v methanol. The Arrhenius plot obtained in 50% v/v methanol is linear over the -30 to -23 degrees C temperature range, and the calculated energy of activation, 8.2 +/- 0.8 kcal/mol, is in good agreement with the value of 7.4 +/- 0.7 kcal/mol found for the reaction in aqueous solution. Collectively, these data indicate that methanol is the best cosolvent for cryoenzymological studies, that ethanol and dimethyl sulfoxide are also suitable cosolvents, and that the presence of any of these cosolvents at either ambient or subzero temperatures does not perturb the catalytic pathway.
D000813:Anilides / Q000378:metabolism*; D000818:Animals; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D011489:Protein Denaturation; D013552:Swine
D000813:Anilides; D000431:Ethanol; C001536:1-leucine-4-nitroanilide; D004126:Dimethylformamide; D007931:Leucyl Aminopeptidase; D000432:Methanol; D004121:Dimethyl Sulfoxide