Purification and Characterization of a Novel Peptidase (IImes) from Mesquite (Prosopis velutina) Pollen

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 (E⅐I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from ␣-chymotrypsin and ␣ 1 -antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of ␣-chymotrypsin with a fluorescent derivative of ␣ 1 -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 E⅐I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1 cleavage.1 is a human serine proteinase inhibitor (serpin), a superfamily of proteins believed to have evolved from a common ancestral gene over ϳ500 million years (1-3). The involvement of ACT in Alzheimer's disease (4, 5) and in the regulation of the inflammatory response (6) 2 as well as of prostate-specific antigen activity (8) makes it a particularly interesting protein for study. As is typical of serpins, ACT (I) forms an enzymatically inactive, 1:1 complex (denoted E*I*) with its target proteinases (for example, chymotrypsin) that releases free enzyme and cleaved ACT (I*) only very slowly (9, 10). The complex is designated E*I* to indicate that conformational change has taken place in both the enzyme (10, 11) and inhibitor (12, 13) moieties. For some serpin-proteinase interactions, full inhibition of proteinase requires Ͼ1 eq of serpin. The ratio of moles of inhibitor required per mole of proteinase for 100% inhibition is defined as the stoichiometry of inhibition (SI). Values of SI Ͼ 1 reflect a partitioning of serpin between inhibitor and substrate pathways, giving rise to "suicide inhibitor"-type kinetics (9, 14 -16).A characteristic feature of the serpin-proteinase complex is that it is stable to both heat and SDS treatment, implying covalent bond formation between enzyme and serpin. The nonlability of E*I* may be due either to distortion of the enzyme active site within the complex (10, 11) or to inaccessibility of the covalent E-I linkage toward attacking nucleophilic water, or both. The position of cleavage in released I* occurs within a so-called "reactive center loop," which in intact I extends out from the rest of the molecule, contains a segment of modified ␣-helix (17-21), and is the primary interaction site between the inhibitor and the target proteinase. Following standard nomenclature (22), the position of cleavage takes place between the P1 and P1Ј sites of the inhibitor, which in ACT corr...

mentioning

confidence: 73%

“…Given the evidence that E*I* may correspond to acyl-enzyme or to the final tetrahedral intermediate (35) resulting from water attack on the acyl-enzyme, an interesting possibility is that the observed cleaved I may arise from the breakdown of the final tetrahedral intermediate during quench/analysis.…”

mentioning

confidence: 99%

The Kinetic Mechanism of Serpin-Proteinase Complex Formation

O’Malley

Nair

Rubin

et al. 1997

“…E*I*s formed from a variety of serpin-serine proteinase pairs are stable to both boiling water and SDS treatment, implying covalent bond formation between enzyme and serpin. Parallel studies by both Lawrence et al (15) and Wilcynska et al (16) have clearly shown that the P1-P1Ј bond is cleaved within the complexes formed by several such pairs (including plasminogen activator inhibitor-1:t-plasminogen activator, plasminogen activator inhibitor-1:u-plasminogen activator, ␣ 1 -proteinase inhibitor:human neutrophil elastase, ␣ 1 -proteinase inhibitor: porcine pancreatic elastase and ␣ 2 -antiplasmin:plasmin), providing strong evidence that E*I* corresponds either to an acyl enzyme, or, less likely given the stability of E*I*, to the tetrahedral intermediate formed by water attack on the acyl enzyme (17).…”

mentioning

confidence: 96%

Antichymotrypsin Interaction with Chymotrypsin

Nair¹,

Cooperman

1998

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1 linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E ؉ I º E ⅐I º EI º E*I* in which EI, which retains the P1-P1 linkage, is formed in a partly or largely ratedetermining step, depending on temperature (O'Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354 -5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1 residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E⅐I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise from EI conversion to E*I*. These results support our earlier conclusion that the P1-P1 linkage is preserved in EI and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, has a rate constant for postcomplex fragment formation from the E⅐I complex very similar to that observed for WT-rACT, implying that EI is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs. Antichymotrypsin (ACT, I)1 is a human serine proteinase inhibitor (serpin), 398 amino acids long, that, as is typical of serpins (1, 2), forms an enzymatically inactive, 1:1 complex (denoted E*I*) with its target proteinases, releasing free enzyme (E) and cleaved ACT (I*) only very slowly (3, 4). The involvement of ACT in Alzheimer's disease (5, 6) and in the regulation of the inflammatory response (7) as well as of prostate-specific antigen activity (8), makes it a particularly interesting protein for study. In the interaction of ACT with chymotrypsin (Chtr) to form an E*I* complex, both proteins undergo extensive conformational change (4, 9); the nonlability of E*I* may be due either to distortion of the enzyme active site within the complex (4, 9) or to inaccessibility of the covalent E-I linkage toward attacking nucleophilic water, or both.Cleavage of I to form released I* occurs between residues 358 and 359 of ACT within the so-called "reactive center loop," which in intact I extends out from the rest of the molecule, contains a segment of modified ␣-helix (10) and is the primary interaction site between the inhibitor and the target proteinase. Following standard nomenclature (11), these residues are de...

“…Early studies favored trapping of the proteinase at the acyl intermediate stage of proteolysis, based on the observation that the serpin reactive center loop was cleaved in the stable complex with proteinase (8, 16 -18). However, NMR evidence later suggested that stabilization of the serpin-proteinase complex at the acyl intermediate stage occurred only under denaturing conditions and that under native conditions, the complex was stabilized at the tetrahedral intermediate stage (19). Yet more recent findings have questioned the NMR data by showing that, even under nondenaturing conditions, the reactive center loop is cleaved concomitant with formation of the serpin-proteinase complex, thus supporting an arrest of the serpin-proteinase reaction at the acyl intermediate stage (20 -22).…”

mentioning

confidence: 99%

Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Olson

Swanson²,

Patston

et al. 1997

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859 -14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and ␤-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, ␣ 1 -proteinase inhibitor (␣ 1 PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a ratelimiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the ␣ 1 PI reactive center P1-P1 bond. Quantitation of kinetic constants for DCI and ␣ 1 PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by ␣ 1 PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.