Use of circular dichroism to study the interactions of carboxypeptidase A (Anson) and A α+β with substrates and inhibitors

Abstract

1. 1. The phenylalanyl circular dichroism (CD) bands of peptides were used to assay peptidase activity of carboxypeptidase A (Anson) and A α+β (70% A α Leu and 30% A β Val). Gly-Gly- l-Phe and Gly- l-Phe have a sharp, negative CD band at 267 nm, whereas l-phenylalanine (the optically active product) has positive CD. Thus the hydrolysis of these substrates may be measured from the CD change at 267 nm (change in Δ ϵM of about 0.04 M −1·cm −1). 2. 2. The addition of β-phenylpropionate to either carboxypeptidase A α+β or A (Anson) makes the CD more positive in the region from 270 to 285 nm. Apparently this alteration results from the tyrosyl CD bands of carboxypeptidase A (peptidyl- l-amino acid hydrolase, EC 3·4.2.1). Evidence is presented that this change arises primarily from the interaction of β-phenylpropionate with Tyr 198 (binding constant, 0.5 mM). 3. 3. Gly- l-Phe does not produce any major alteration of the tyrosyl CD bands of carboxypeptidase A α+β or A (Anson). Apparently the movement of Tyr 248 into the active site does not cause any readily measurable CD alteration, even when extensive signal averaging is carried out to achieve low noise records (peak-to-peak noise less than 5·10 −6 ΔA). 4. 4. The binding of Gly- l-Phe, Gly- d-Phe, l-phenylalanine, or d-phenylalanine shifts the wavelength positions of the tryptophanyl CD fine structure observed in carboxypeptidase A (Anson) cooled to −196°. This effect may result from binding outside the active site pocket, possibly in the groove near Arg 71.