Evidence for a structural change in the substrate preceding hydrolysis of a chymotrypsin acyl enzyme: Application of the resonance Raman labelling technique to a dynamic biochemical system

Abstract

By using a flow system, resonance Raman spectra have been obtained of the acyl enzyme 4-amino-3-nitro-cinnamoyl-α-chymotrypsin over the pH range where it shifts from a stable to an active state. The band at 1625 cm −1 was assigned to the CC stretch in the acryloyl residue ▪ and bands in the 1200 to 1450 cm −1 region were shown to be associated with the aromatic residue. Studies with analogues of the substrate established that the CC and the aromatic bands could independently monitor events in the ▪ and aromatic moieties, respectively. Thus, events in the acyl enzyme leading to spectral changes involving the catalytic site could be distinguished from those involving the aromatic site. The data strongly suggested that the structure of the acylating group in the stable acyl enzyme at pH 3.0 resembled that of the substrate in aqueous solution, the conformation about the ethylenic bonds being essentially planar, trans and probably s- trans about the CCCO single bond. However, on shifting the acyl enzyme to pH values of 5.7 to 7.0 a large change occurred in the 1625 cm −1 band without detectable changes in the aromatic bands. Thus, the enzyme produces a change in a submolecular grouping which contains the bond being hydrolysed. The change occurred within the mixing and observation time of about ten seconds. Since it was identical at essentially inactive (pH 5.7) and active (pH 7.0) pH values it must precede and be independent of the rate-controlling step in deacylation. The change in the 1625 cm −1 band thus probably reflects an ionization in the acyl enzyme between pH 3.0 and 5.7. Consequently, above pH 5.7 deacylation proceeds from a structure differing from that at the stable pH of 3.0. The resonance Raman spectra of this intermediate are consistent with either deformation to reduce planarity within the acryloyl residue, or of the stabilization of resonance structures of the form ▪ Either of these mechanisms would produce an intermediate, the carbonyl group of which would be probably intrinsically more reactive relative to the substrate in solution or to the stable acyl enzyme. The resonance Raman provides some support for Henderson's (1970) prediction that distortion occurs in the acryloyl residue prior to deacylation.