PHOTOCHEMISTRY OF ENZYME-BOUND CINNAMOYL DERIVATIVES

p-(Diethylamino)-o-hydroxy-alpha-methylcinnamoyl (CINN) was used as an acylating moiety for the formation of stable CINN-enzymes at the acti ve site serine residues of the enzymes chymotrypsin, Factor Xa, and th rombin. Photolysis of the stable CINN-enzymes generates enzymatic acti vity via the proposed consecutive steps of photoisomerization (rate-de termining step) and thermal lactonization (fast). Photochemical studie s were undertaken to assess how an enzyme active site could alter the photochemistry of the cinnamoyl derivative. Quantum yields for E to Z photoisomerization (PHI(E-->Z)) were measured for the three CINN-enzym es at 366 nm (20-degrees-C in pH 7.4 Tris buffer) and for the model sy stem ethyl p-(diethylamino)-o-hydroxy-alpha-methylcinnamate (CINN-OEt) . Relative to the value of PHI(E-->Z) = 0.13 for CINN-OEt, CINN actual ly displayed an enhanced isomerization efficiency while bound at the a ctive sites of chymotrypsin and Factor Xa (PHI(E-->Z) = 0.17 and 0.23, respectively) and a decreased isomerization efficiency (PHI(E-->Z) = 0.04) while bound to thrombin. The influence of chymotrypsin's active site on cinnamoyl photoisomerization was investigated further by measu ring the photostationary state isomeric ratios for MeCINN-chymotrypsin and MeCINN-OEt, the methyl ether analogue of the corresponding CINN p hotolytes, in pH 7.4 Tris buffer. MeCINN-chymotrypsin displayed a valu e of 2.7 for PHI(Z-->E)/(PHI(E-->Z = quantum yield for E to Z and PHI( Z-->E) = quantum yield for Z to E), and MeCINN-OEt exhibited a value o f 1.7. This difference could not be attributed to the greater hydropho bicity of chymotrypsin's active site since values of PHI(Z-->E/PHI(E-- >Z) for MeCINN-OEt in organic solvents were less than unity. Photoisom erization quantum yields were also measured for the isomers of MeCINN- chymotrypsin and MeCINN-OEt. Both acyl-enzyme isomers exhibited less e fficient photoisomerization (E to Z and Z to E) than their respective model system, MeCINN-OEt.