ENDOR DETERMINED STRUCTURE OF A COMPLEX OF ALPHA-CHYMOTRYPSIN WITH A SPIN-LABELED TRANSITION-STATE INHIBITOR ANALOG

The conformation of the spin-labeled compound ethyl-1-oxypyrrolinyl-3- carboxyl)-L-phenylalaninal (SLPheal), synthesized as a transition-stat e inhibitor analogue of alpha-chymotrypsin (alpha CT), has been determ ined by electron nuclear double resonance (ENDOR) and molecular modeli ng methods for both the free inhibitor in solution and the hemiacetal enzyme:inhibitor adduct. SLPheal exhibited linear, competitive inhibit ion of alpha CT with K-I = (0.21 +/- 0.03) x 10(-3) M. By a combinatio n of NMR and ENDOR spectroscopy, we have established that 92% of the a ldehyde is found as the hydrated aldehyde species RCH(OH)(2) at pH 7.0 . From ENDOR spectra, principal hyperfine coupling (hfc) components of specific protons assigned by selective deuteration were determined, a nd their corresponding dipolar hfc components were computed to estimat e electron-proton distances. The principal hfc components of the fluor ine substituent of a zeta F-phenylalaninal inhibitor analogue were als o determined for estimation of electron-fluorine distances. With these ENDOR determined distances as constraints, the conformation of the in hibitor both free in solution and in the active site of alpha CT was d etermined on the basis of torsion angle search calculations and molecu lar graphics analysis. Comparison of the conformation of the inhibitor free in solution and bound to alpha CT showed that formation of the h emiacetal enzyme:inhibitor adduct required positioning of the spin-lab el group with torsional alteration in inhibitor structure similar to t hat described for the spin-labeled tryptophanyl moiety in an acylenzym e reaction intermediate of alpha CT [Wells et al., J. Biol. Chem. 1994 , 269, 4577-4586]. While the ENDOR results of the hemiacetal formed in neat methanol were distinguishably different for R and S configuratio ns modeled for an sp(3) hybridized C atom of the aldehyde functional g roup, only an S configuration in the enzyme:inhibitor adduct was compa tible with all of the ENDOR constraints. Since the inhibitor in aqueou s solution is found largely in its hydrated form, it is probable that this is the predominant species that is initially bound in the active site of the enzyme. Accordingly, a reaction scheme is suggested for de hydration of the active site bound diol species catalyzed by the proto nated form of His-57 to allow formation of the hemiacetal adduct with the side chain of Ser-195 having only an S configuration.