Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A

Abstract

The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH 2CO 2CH(CH 2CH 3)CO 2H ( 2 ) by bovine pancreatic carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.12.2) have been studied over the substrate concentration range 10 −4–10 −1 M at pH 7.5, 25°C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH 3, CH 3CH 2 and (CH 3) 3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES 2 complex and parameters for the second-degree rate equation v/ E = ( k 2 app S + k 3 app S 2/ K SS app )/( K S app + S + S 2/ K SS app ) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH 3 and Y = cyclo-C 6H 11 the following linear correlations were observed: -log K S app = 0.82 π + 1.32 and log k 2 app/ K S app = 0.71 π + 5.81 (π is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. K SS app also depends on the hydrophobicity of Y but no quantitative correlation is obvious. Thus the Y unit of 2 is involved in a hydrophobic interaction with the enzyme when 2 binds at both the catalytically productive and inhibitory sites. Parameters for the enzymic hydrolysis of the esters YCONHCH 2CO 2CH(CH 2CH(CH 3) 2)CO 2H ( 3 ) (Y = C 6H 5(CH 2) n ( n = 0, 1, 2)) are also presented. Pronounced non-productive 1:1 enzyme · substrate complex formation is observed for each of 2 : Y = C 6H 5(CH 2) n ( n = 2,3) and 3 : Y = C 6H 5(CH 2) 2. Hippurate anion is shown to be an uncompetitive inhibitor ( K i = 12 mM) for the hydrolysis of 2 : Y = (CH 3) 3C. Data are now available which can only be interpreted in terms of at least three enzymic sites being available for hydrophobic interactions with ester substrate molecules.