Interaction of carboxypeptidase A with carbamate and carbonate esters

Abstract

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.

Bioreversible Derivatives of Phenol. 2. Reactivity of Carbonate Esters with Fatty Acid-like Structures Towards Hydrolysis in Aqueous Solutions

“…The carbonate esters with fatty acid-like structure were synthesized from phenyl chloroformate and the appropriate ω-hydroxycarboxylic acids or ethyl ester of the ω-hydroxycarboxylic acids, essentially following the procedures described by King et al [36] (see Experimental section for details). In addition to the carbonate esters with fatty acid-like structure listed in Table 1, attempts were made to synthesize 4-(phenoxycarbonyloxy)-butyric acid.…”

“…The compounds were synthesized by procedures modified from those reported by King et al [36]. HPLC was used to monitor progress of reactions.…”

Metal Ion Catalysis in the Hydrolysis of Esters of 2-Hydroxy-1,10-phenanthroline: The Effects of Metal Ions on Intramolecular Carboxyl Group Participation

“…The pH-independent value of k cat in the enzymatic reaction (195 s Ϫ1 at 30 ЊC) (16) is only ϳ500-fold larger than k 2 Ј with III and IV. However, in contrast with the excellent leaving group of III and IV, the leaving group alcohol of the ␤-phenyllactate ester is of much higher pK a ; that pK a was calculated to be 14.7 (40). Therefore, the enzymatic reaction is considerably more favorable than the metal ion-promoted carboxyl nucleophilic reactions of III and IV.…”

Inhibition Effects in the Hydrolysis Reactions of Esters and Peptides Catalyzed by Carboxypeptidase A: An Example of Cooperative Binding Effects with a Monomeric Enzyme

“…There is strong evidence that peptides and esters bind initially to CPA in different sites (7,8). Indole-3-acetic acid (IAA) is a competitive inhibitor toward ester substrates but is noncompetitive toward peptides (7).…”

“…Indole-3-acetic acid (IAA) is a competitive inhibitor toward ester substrates but is noncompetitive toward peptides (7). The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine is a linear competitive inhibitor toward the peptide hippuryl-L-phenylalanine but is a linear noncompetitive inhibitor toward the ester hippuryl-L,β-phenyllactate (8). The carbamate ester is also an observable substrate for the enzyme at high enzyme concentration (10 −6 M), and K m = K i .…”