Formation of the covalent chymotrypsin.antichymotrypsin complex involves no large-scale movement of the enzyme.

alpha(1)-Antichymotrypsin is a member of the serine proteinase inhibitor, or serpin, family that typically forms very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*) with its target proteinases. Serpins share a conserved tertiary structure, in which an exposed region of amino acid residues (called the reactive center loop or RCL) acts as bait for a target proteinase. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser(195) of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta-sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha(1)-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E.I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.

The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex.

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.

Heart rate and metabolic response to competitive squash in veteran players: identification of risk factors for sudden cardiac death.

We studied 10 older males during a competitive game and the early post-exercise period to define the metabolic response to squash in veteran players. For comparison, all subjects were also studied during exhaustive treadmill exercise. Squash caused a dramatic increase in heart rate (150%), and circulating levels of noradrenaline (164%), adrenaline (93%), lactate (202%) and free fatty acids (67%). These effects were independent of haemoconcentration. The early post-exercise period (5 min) was characterized by persistent elevation of plasma catecholamines, lactate, and free fatty acids, hypokalaemia and ventricular arrhythmias. The heart rate and metabolic responses to squash were similar in pattern and magnitude to those observed during treadmill exercise, highlighting the strenuous nature of squash as a recreation sport. While these changes may represent appropriate physiological adaptation to exercise in health, each has been implicated in the pathogenesis of fatal ventricular arrhythmias in subjects with ischaemic heart disease. These data support the contention that squash may be an inappropriate form of exercise for older men with coronary artery disease.