Two extremely potent inhibitors of citrate synthase, carboxyl and primary amide analogues of acetyl coenzyme A, have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.70- and 1.65-A resolution, respectively. The inhibitors have dissociation constants in the nanomolar range, with the carboxyl analogue binding more tightly (Ki = 1.6 nM at pH 6.0) than the amide analogue (28 nM), despite the unfavorable requirement for proton uptake by the former. The carboxyl group forms a shorter hydrogen bond with the catalytic Asp 375 (distance < 2.4 A) than does the amide group (distance approximately 2.5 A). Particularly with the carboxylate inhibitor, the very short hydrogen bond distances measured suggest a low barrier or short strong hydrogen bond. However, the binding constants differ by only a factor of 20 at pH 6.0, corresponding to an increase in binding energy for the carboxyl analogue on the enzyme of about 2 kcal/mol more than the amide analogue, much less than has been proposed for short strong hydrogen bonds based on gas phase measurements [> 20 kcal/mol (Gerlt & Gassman, 1993a,b)]. The inhibitor complexes support proposals that Asp 375 and His 274 work in concert to form an enolized form of acetyl-coenzyme A as the first step in the reaction.

The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.

Computational studies were performed in an effort to understand the relative reactivity of oxoesters and thioesters in nucleophilic acyl transfer reactions. Transition state models were developed for the reactions of methyl acetate and methyl thioacetate with hydroxide, ammonia, and methylcyanoacetate carbanion. Quantum mechanical calculations based on these models reproduced experimental observations that oxoesters and thioesters have similar reactivity toward hydroxide while thioesters are about 100-fold and at least 2000-fold more reactive than oxoesters toward amine and carbanion nucleophiles, respectively. NBO analysis was performed to elucidate the role of electron delocalization in reactant and transition state stabilization. These calculations indicate similar losses of delocalization energy for the oxoester and thioester in going from the reactants to the transition state in reaction with hydroxide while the loss of delocalization energy is significantly greater for the oxoester in reactions with the other nucleophiles. Bond rotational analysis of the transition states for the reactions with hydroxide and ammonia provide support for an important role of the p(X) --> sigma(C-Nu) interaction (X = O or S of the oxoester or thioester respectively, Nu = nucleophile) in governing the reactivity of oxoesters and thioesters in nucleophilic acyl substitution.

E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

Transition state structures and energies have been investigated for concerted and stepwise mechanisms for the acyltransfer reactions of ethyl acetate and ethyl thioacetate with ammonia. Specific and general solvent effects have been evaluated. The results predict stepwise mechanisms involving water-catalyzed proton transfer for both reactions and indicate that the thioester is more reactive than the oxoester in both the addition and elimination steps.

An improved method for the synthesis of analogs of coenzyme A (CoA) and its thioesters, which are modified in the thiol or thioester moiety, has been developed using a combination of chemical and enzymatic reactions. The enzymes catalyzing the last two steps of CoA biosynthesis were used to prepare a CoA analog (lc) in which an amide bond is replaced by a thioester bond and the thiol group is replaced by a methyl group. Reaction of lc with a primary amine in aqueous solution results in aminolysis of the thioester linkage to form the desired CoA analog. Reaction with different amines permits the introduction of a variety of functional groups in place of the normal thiol or thioester group. This methodology has been used in the synthesis of five new analogs of acetyl-CoA in which the thioester sulfur is replaced by a methylene group and the acetyl group is replaced by carboxylate (14a), nitro (14b), carboxamide (14c), methyl sulfoxide (14d), and methyl sulfone (14e) groups. 14a-c were designed to mimic the possible enolate or enol intermediate in the reaction of citrate synthase and related enzymes. 14a and 14c are potent inhibitors of citrate synthase, with K-, values 1000-and 570-fold lower than the Km for acetyl-CoA, respectively. CD titrations indicate that 14a and 14c have low affinity for citrate synthase in the absence of oxaloacetate, consistent with their recognition as enol or enolate analogs. 14b is a poor inhibitor of citrate synthase, with affinity slightly lower than that for acetyl-CoA. These results are consistent with generation of the enol form of acetyl-CoA as the nucleophilic intermediate in the reaction of citrate synthase. 14d and 14e were designed to mimic the tetrahedral intermediate or transition state in the reaction of chloramphenicol acetyltransferase and related acetyl-CoA-dependent acetyltransferases. Both compounds are poor inhibitors of chloramphenicol acetyltransferase, with affinities slightly lower than that of acetyl-CoA, indicating that these compounds are not good mimics of the enzyme-bound tetrahedral intermediate or transition state.

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