The theory and application of transition state pK(a) values: the reaction of papain with a series of trimethylene disulphide reactivity probes

For many years methods to describe the structure of the transition state for a reaction have been sought. Most commonly these structures have been inferred from kinetic isotope effects. We report here for the first time the application of transition state pK(a) values to describe the relationship between molecular recognition and the transition state for the catalytic mechanism of papain. The background to the theory is presented and applied to the reactions of papain with a series of trimethylene disulphide reactivity probes. The common feature of these reactions is a loss in reactivity on ionization of the imidazolium cation for those probes containing molecular recognition features and an increase in reactivity on ionization of the electrostatic switch residue. The use of transition state pK(a) values enhances this information by providing details regarding the protonic distribution within the transition state. This has led to the reconsideration of the effect of the electrostatic switch ionization and the role of the hydrogen bond formed between the catalytic-site imidazolium cation and the leaving group of the reaction in the catalytic mechanism of papain.     

Regulation of the lipopolysaccharide-specific sialyltransferase activity of gonococci by the growth state of the bacteria, but not by carbon source, catabolite repression or oxygen supply

The enzyme sialyltransferase (STase) of Neisseria gonorrhoeae is a major pathogenicitiy determinant. Using a refined method for assaying the STase activity, the Km for CMP-NANA was shown to be 14 +/– 2 M, higher than that reported previously. Rates of sialylation by Nonidet extracts, prepared under conditions that optimise solubilisation of the membrane-bound enzyme, were 6 to 20 nmol of NANA transferred from CMP-¹⁴C-NANA onto isolated lipopolysaccharide/min./mg of extracted protein, far higher than the previously reported rates of less than 1 nmol of NANA transferred/min./mg of extracted protein. Gonococci grew more slowly with lactate or pyruvate than with glucose as the carbon source. Although growth with a mixture of limiting concentrations of both glucose and lactate was biphasic, diauxic growth was also found in the control culture supplied with glucose alone. The growth rate in the presence of lactate alone was slower than with glucose. The growth rate increased slightly relative to the glucose culture when both substrates were available; lactate was consumed more rapidly than glucose. Higher STase activities were found in bacteria harvested in the exponential than in the stationary phase of aerobic growth: the activity in aerated cultures was higher than those of oxygen-limited or anaerobic cultures. Similar STase activities were found in bacteria that had been grown with glucose, lactate or pyruvate as the carbon and energy source. Sialyltransferase synthesis is essentially constitutive: it is not regulated by glucose repression or by induction by lactate or anaerobiosis.     

Characterization of a conformational epitope on hepatitis B virus core antigen and quasiequivalent variations in antibody binding

We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to approximately 10-A resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides-two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid-T=3 and T=4 particles-present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from approximately 100 to approximately 0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.     

Novel cell-permeable acyloxymethylketone inhibitors of asparaginyl endopeptidase

Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently identified lysosomal cysteine protease belonging to clan CD. To date it has been shown to be involved in antigen presentation within class II MHC positive cells and in pro-protein processing. Further elucidation of the biological functions of the enzyme will require potent and selective inhibitors and thus we describe here new acyloxymethylketone inhibitors of AEP. The most potent of the series is 2,6-dimethyl-benzoic acid 3-benzyloxycarbonylamino-4-carbamoyl-2-oxo-butyl ester (MV026630) with a kobs/[I] value of 1.09 x 10(5) M(-1) s(-1). At low microM concentrations this compound is able to enter living cells and irreversibly inactivate AEP. We show that this results in inhibition of AEP autoactivation and in perturbation of the processing and presentation of T cell epitopes from both tetanus toxin and myelin basic protein.