Spin-lattice and transverse fluorine relaxation rates have been determined for the title enzyme derivative at pH 7. The data have been analyzed to provide an estimate of the rotational correlation time (rc) near the trifluoromethylbenzenesulfonyl group and the correlation time (r¡) for internal rotation of trifluoromethyl. The major part of the fluorine relaxation is due to proton-fluorine dipole-dipole interactions. Specific deuteration experiments show that these interactions predominantly involve protons of the enzyme and solvent.

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We recently reported a study of the complexes formed between p-fluorocinnamate (1) and a-chymotrypsin;' 1 is an effective competitive inhibitor of enzymic action and thus is likely to bind at or very near the active site of the protein. Chymotrypsin is a highly selfassociating protein2 and our results suggested that 1 binds to the monomeric, dimeric and trimeric forms of the enzyme equivalently or nearly equivalently both in the thermodynamic sense, as reflected by the dissociation constant for each complex, and also in terms of fluorine NMR parameters (chemical shifts and relaxation rates). The monomer has one binding site while the dimer exhibits two and the trimer, three. The relaxation rate data also showed that a substantial part of fluorine-proton dipole-dipole interactions present in the enzymesmall molecule complexes involve interactions with protons of the macromolecule. coocoocoo-Unlike p-fluorocinnamate, o -and rn -fluorocinnamate (2 and 3) can exist as two conformational isomers (a and b) generated by rotation about the single bond between the aromatic ring and the acrylate group. It was of interest to determine the extent to which each of these conformers is bound to chymotrypsin. The enzyme-bound form of a small molecule need not have the same conformation that the molecule takes up when free in solution if the * Author to whom correspondence should be addressed.binding of a non-ground state conformation offers an advantage in the catalytic mechanism. The methods of the previous study have been applied to examination of the complexes formed between 2 and 3 and achymotrypsin, and we describe here the results of these experiments. EXPERIMENTALMaterials. o -Fluoro-and rn -fluorocinnamic acids were synthesized as described ear lie^.^ a -Deuterio-ofluorocinnamic acid and p -deuterio-o -fluorocinnamic acid were the same samples as used previously? Buffers, enzyme substrate and native enzyme were identical to those of the prior study.' Enzyme active site titrations employed trans-cinnamoylimidazole (5X re~rystallized)~ and consistently showed 75-82'/0 active sites from batch to batch of enzyme. All enzyme concentrations were expressed as active site concentrations. Procedures. All instrumentation was identical to that used in the earlier studies of the p-fluorocinnamate complexes. Fluorine NMR samples were prepared as described previously and the enzyme kinetic studies and binding constant determinations were executed according to the earlier procedures.' RESULTS Kinetic inhibition studies.Inhibition of the chymotryptic hydrolysis of glutarylphenylalanine-p-nitroanilide by o-fluoro-and rn-fluorocinnamate was examined.',6 Lineweaver-Burke kinetics were observed and both 2 and 3 appeared to be competitive inhibitors of the enzyme. Dissociation constants (KJ were computed from the rate data and are presented in Table 1.

SynopsisFluorine nmr experiments carried out at 51.0 and 94.1 MHz have been used to explore the interaction of the probe molecule p-fluorocinnamate with conjugates formed from a-chymotrypsin and poly(N-acryloyl-@-alanine). The data obtained include enzyme-induced chemical-shift effects, spin-lattice (R1) and transverse (Rz) relaxation rates, and the rate constant for dissociation of the fluorocinnamate+nzyme complexes. Analysis of the results indicates that while overall molecular tumbling of the enzyme molecule is not greatly changed by attachment of polymers of various sizes, conjugated polymer can appreciably affect the structure of the p-fluorocinnamate binding site. The important variable involved in such structural changes appears to be the amount of polymer present per mole of protein.

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