Alpha-Methyl substrates of carboxypeptidase A. A steric probe of the active site.

Abstract

Although optical resolution of alpha-methylphenylalanine (alpha-Me-Phe) has been achieved by the action of carboxypeptidase A on the N-trifluoroacetyl derivative of the amino acid (TFA-alpha-Me-Phe), it is improbable that an alpha-methyl substrate could bind in the same orientation as glycyl-L-tyrosine, due to steric interaction of the alpha-methyl group with an atom in the imidazole ring of zinc ligand His-196. The kinetic parameters for TFA-alpha-Me-Phe and for an ester substrate bearing an alpha-methyl group (beta-hippuryl-alpha-methylphenyllactic acid, HMPL) have been determined and compared to those for the appropriate nonmethylated control substrates. Both TFA-alpha-Me-Phe and HMPL appear to be bound nearly as well as are their respective controls, and HMPL is hydrolyzed nearly as rapidly as its control. TFA-alpha-Me-Phe, however, is hydrolyzed only about one-fiftieth as rapidly as is the nonmethylated substrate. These findings are consistent with the possibilities that: (1) the proposed substrate-induced conformational shift of Tyr-248 is hindered when the methylated substrates are bound; (2) the orientation in which alpha-methyl substrates are bound precludes optimal positioning of Tyr-248 and the scissile bond even after the rotation of Tyr-248 has occurred; (3) amides and esters are bound in different orientations, and in the amide orientation an alpha-methyl group is so directed as to interfere with the approach of Glu-270 to the scissile bond.