Investigations of the chymotrypsin-catalyzed hydrolysis of specific substrates. V. Determination of pre-steady state kinetic parameters for specific substrate esters by stopped flow techniques.

摘要: Abstract It has been suggested that the chymotrypsin-catalyzed hydrolysis of specific substrates is described by a three-step mechanism: [see PDF for equation] and esters k23 much greater than k34, K's steady state kinetic parameter Km(app). Steady investigations do not allow one to determine individual parameters mechanism shown above. This information can be obtained from pre-steady investigations. In this paper we are reporting parameters, determined stopped flow techniques, hydrolyses ethyl N-acetyl-l-tyrosine, together with previously published data N-acetyl-l-tryptophan N-acetyl-l-phenylalanine. allows comparison K's, k23, k34 catalyzed all three aromatic amino acids which considered chymotrypsin. Also included an investigation presteady pertaining N-acetyl-l-leucine methyl ester amide tosyl-l-arginine. N-Acetyl-l-leucine was chosen because it pH dependence study possible acids, since in these reactions becomes too large above 6 adequately measured technique. The found similar k34. available previously. p-Tosyl-l-arginine N-α-p-tosyl-l-argininamide were because, as typical trypsin hydrolyzed chymotrypsin also, they expected offer some insight into specificity reaction. Unlike substrate esters, accumulation intermediate, such EP2 equation, could detected. One obvious explanation observation rather rate-limiting. unproductive binding unspecific also account data.