1. Dependence of the catalytic activity of papain on the ionization of two acidic groups
S D Lewis, F A Johnson, A K Ohno, J A Shafer J Biol Chem. 1978 Jul 25;253(14):5080-6.
The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.
3. [Structure of the active center of subtilisin 72]
N F Kazanskaia, O A Kost Biokhimiia. 1982 May;47(5):834-41.
The inhibition by N-benzoyl-L-arginine of subtilisin-catalyzed hydrolysis of various substrates was investigated. Study of combined hydrolysis of these substrates revealed the existence of two productive binding sites in the subtilisin 72 molecule. The type of substrate adsorption depends on the nature of the acyl moiety of the molecule and on the nature of the split-off group. The N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-citrulline methyl ester are bound at the same adsorption site (A), while N-cinnamoyl imidazole and N-acetyl-L-valine methyl ester are found at another adsorption site (B); N-benzoyl-L-alanine methyl ester can be adsorbed both at site A and at site B. However, substitution of the split-off group in the substrates previously adsorbed at site B by the p-nitrophenyl group causes their transfer to site A.