The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-alpha PGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-alpha PGA. The X-ray crystal structure (2.0-A resolution) of MR inactivated by (R)-alpha PGA revealed the presence of a covalent adduct formed by nucleophilic attack of the epsilon-amino group of Lys 166 on the distal carbon on the epoxide ring of (R)-alpha PGA. The proximity of the alpha-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-alpha PGA] was corroborated by the crystal structure (2.1-A resolution) of MR complexed with the substrate analog/competitive inhibitor, (S)-atrolactate [(S)-alpha-methylmandelate]. These results support the proposal that Lys 166 is the polyvalent acid/base responsible for proton transfers on the (S) face of mandelate. In addition, the high-resolution structures also provide insight into the probable interactions of mandelate with the essential Mg2+ and functional groups in the active site.

Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.

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