α-Methyl Substrates of Carboxypeptidase A. Steric Probe of the Active Site

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Abstract

Although optical resolution of α-methylphenylalanine (α-Me-Phe) has been achieved by the action of carboxypeptidase A on the N-trifluoroacetyl derivative of the amino acid (TFA-α-Me-Phe), it is improbable that an α-methyl substrate could bind in the same orientation as glycyl-l-tyrosine, due to steric interaction of the α-methyl group with an atom in the imidazole ring of zinc ligand His- 196. The kinetic parameters for TFA-α-Me-Phe and for an ester substrate bearing an α-methyl group (β-hippuryl-α-methylphenyllactic acid, HMPL) have been determined and compared to those for the appropriate nonmethylated control substrates. Both TFA-α-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-α-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 α-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 α-methyl group is so directed as to interfere with the approach of Glu-270 to the scissile bond.

Original languageEnglish
Pages (from-to)2631-2635
Number of pages5
JournalBiochemistry
Volume14
Issue number12
DOIs
StatePublished - Jun 1 1975

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