Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A.
The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH2CO2CH(CH2CH3)CO2H (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 degrees C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH3, CH3CH2 and (CH3)3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES2 complex and parameters for the second-degree rate equation v/E = (kapp2 S + kapp3 S2/KappSS)/(KappS + S + S2/KappSS) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH3 and Y = cyclo-C6H11 the following linear correlations were observed: -log KappS = 0.82 pi + 1.32 and log kapp2/KappS = 0.71 pi + 5.81 (pi is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. KappSS 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 inhibitor sites. Parameters for the enzymic hydrolysis of the esters YCONHCH2CO2CH(CH2CH(CH3)2)CO2H (3) (Y = C6H5(CH2)n (n = 0, 1, 2)) are also presented. Pronounced nonproductive 1: 1 enzyme.substrate complex formation is observed for each of 2: Y = C6H5(CH2)n (n = 2, 3) and 3: Y = C6H5(CH2)2. Hippurate anion is shown to be an uncompetitive inhibitor (Ki = 12 mM) for the hydrolysis of 2: Y = (CH3)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.
D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D002417:Cattle; D005998:Glycine / Q000031:analogs & derivatives*; D007700:Kinetics; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C016955:N-acylglycine esters; D002268:Carboxypeptidases; D005998:Glycine