Spin label investigations of α-chymotrypsin active site structure in single crystals

Abstract

α-Chymotrypsin single crystals were acylated at Ser195 with R(+)- and S(−)-3-carboxy-2,2,5, 5-tetramethylpyrrolidinyl-1-oxyl p-nitrophenyl ester ( I), respectively, at pH 4.2. The R(+)- and S(−)-acyl nitroxide groups ( II) each took up single unique orientations with respect to the enzyme. Studies of the spin-labeled crystals based on the atomic co-ordinates of the tosyl enzyme and the orientations determined by single crystal electron spin resonance measurements showed that in each case the acyl group was positioned just outside the hydrophobic pocket with the pyrrolidine ring roughly coplanar with the “slit” of the pocket. The R(+)-acyl group was slightly farther away from the pocket and closer to Tyr146′ of the dyad-related molecule than was the S(−)-acyl group. In addition, the carbonyl group of the R(+)-acyl nitroxide moiety appeared to bo positioned more favorably for backside attack by His57 ( via an activated water molecule) than in the case of the S(−)-acyl moiety. This also appeared to be consistent with the R(−)-acyl group deacylating more rapidly than the S(−)-acyl group, both in solution and in the crystalline state. Furthermore, from electron spin resonance studies with these two acyl spin labels, the existence of a non-crystallographic dyad along a ∗ and the monoclinic P 21 2-fold element along b were displayed without requisite X-ray crystallographic symmetry determination, thus demonstrating the electron spin resonance single crystal approach as a valid structural probe of molecular symmetry.