Steady-State Kinetics of Aminopeptidase Catalysis: A Stopped-Flow Radiationless Energy Transfer Study

Stopped-flow radiationless energy transfer experiments have been carried out to investigate the hydrolysis of some dansyl peptide substrates (S) catalyzed by aminopeptidase (E). RET between enzyme tryptophanyl residues and the dansyl group in the substrate allowed direct observation and quantitation of the enzyme-substrate (ES) complexes. Analysis of the stopped-flow RET traces gives k_cat = 1.32 s^?1 and K_M = 47 μM for Leu-Ala-NH(CH₂)₂NH-Dns (Leu-Ala-DED) and k_cat = 4.80 s^?1 and K_M = 196 μM for Leu-Gly-NH(CH₂)₂NH-Dns (Leu-Gly-DED). The activation energies of the enzymatic reactions were determined from the Arrhenius plots to be 57 and 38 kJ mol^?1 for Leu-Ala-DED and Leu-Gly-DED, respectively. The kinetic results indicate that the enzyme binds Leu-Ala-DED more tightly than Leu-Gly-DED as revealed by a small value of K_M. That this enzyme catalyzes the turnover of Leu-Gly-DED more efficiently than Leu-Ala-DED is reflected in a large value of k_cat and a small activation energy. The RET signals during the hydrolysis of Leu-Val-NH(CH₂)₂NH-Dns were extremely weak probably because of the inefficient energy transfer in the ES complex or the retention of the product in the enzyme after completion of the reaction. Aminopeptidase was inactive towards the dansyl compounds of the single amino acid studied. This fact may be due to an unfavorable conformation of these compounds in the ES complexes (small k_cat) or a weak binding of the substrates to the enzyme (large K_M) or both.