Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded .alpha.-chymotrypsin

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Abstract
Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease .alpha.-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values. This change is strikingly similar to that obtained in going from the model aldehyde to the corresponding ester. This leads us to the conclusion that the native acyl enzyme is more "aldehyde-like". We conclude that the environment in the active site of the native protein induces a conformational change in the bound substrate that is responsible for a change in the spectroscopic properties and for the catalytic activity of the enzyme. It is suggested that the observed spectroscopic properties are consistent with a change in the all-planar sp2 structure of the acyl serine ester.