Comparison of MeO-Suc-Val-Pro-Phe-CO2Me (29) and MeO-Suc-Ala-Ala-Pro-Phe- CO2Me (25) with their corresponding trifluoromethyl ketones 9a and 9b, respectively, in rat and human neutrophil cathepsin G assays showed the alpha-keto esters to be more potent inhibitors. Likewise, Ac-Pro-Ala-Pro-Ala-CO2Me (21) was more potent than its corresponding trifluoromethyl ketone (9c) in both porcine pancreatic elastase and human neutrophil elastase assays. Within a set of Ala-Ala-Pro-Val-CF3 elastase inhibitors, the carbobenzyloxy (Cbz) N-protecting group conferred greater potency as a P5 site recognition unit for elastase than did dansyl, methoxysuccinyl, or tert-butyloxycarbonyl. Initial inhibition of elastase was greater when trifluoromethyl ketone 9f was added from a stock solution of dimethyl sulfoxide than when it had been buffer-equilibrated prior to assay, which suggests that the nonhydrated ketone is the more effective form of the inhibitor. The most potent elastase inhibitor we report is Na-(Ad-SO2)-N epsilon-(MeO-Suc)Lys-Pro-Val-CF3 (16) which has a Ki of 0.58 nM.
The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.
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