Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones

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Abstract
Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones (1-4). Ynenol lactones that are .alpha.-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted .alpha. to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25.degree. C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 .+-. 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivative the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R. W., Tam, T. F., Thomas, E. M., Robinson, V. J., and Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597].