An Ester Bond Linking a Fragment of a Serine Proteinase to Its Serpin Inhibitor

Serpins inhibit proteinases through a complicated multistep mechanism. The precise nature of these steps and the order by which they occur are still debated. We compared the fate of active and S195A inactive rat trypsin upon binding to ␣ 1 -antitrypsin and P 1 -Arg-antichymotrypsin using stopped-flow kinetics with fluorescence resonance energy transfer detection and timeresolved fluorescence resonance energy transfer. We show that inhibition of active trypsin by these serpins leads to two irreversible complexes, one being compatible with the full insertion of the serpin-reactive site loop but not the other one. Binding of inactive trypsin to serpins triggers a large multistep reversible rearrangement leading to the migration of the proteinase to an intermediate position. Binding of inactive trypsin, unlike that of active trypsin, does not perturb the rhodamine fluorescence at position 150 on the helix F of the serpin. Thus, inactive proteinases do not migrate past helix F and do not trigger full serpin loop insertion.Serpins are a family of mainly serine proteinase inhibitors, although some members also inhibit cysteine proteinases whereas others have evolved into non-inhibitory forms such as hormone carriers and immunomodulatory factors (1). Serpins have roles in many regulatory processes including fibrinolysis, complement activation, and blood coagulation. The human genome contains more serpins than other classes of serine proteinase inhibitors (2). Whereas canonical inhibitors such as BPTI 1 form non-covalent reversible complexes with their target proteinases, serpins operate through a complex multistep pathway leading to an irreversible covalent adduct. The crystal structure of one of these complexes (3) shows that the reactive site loop of the serpin has been cleaved at the P1-PЈ1 position and has inserted into the A ␤-sheet in a conformation close to that obtained by cleaving the loop with a non-inhibited proteinase. This 70-Å translocation of P1 has dragged the proteinase to the opposite pole of the serpin. The tertiary structure of the complex also confirms that the proteinase forms a covalent acyl-enzyme with the P1 residue of the inhibitor (4 -6) and that the catalytic triad of the enzyme is distorted (7). Recently, the three-dimensional structure of a complex between S195A trypsin and an active site loop modified insect serpin has been solved (8). The position of the proteinase on the top of the uncleaved and uninserted loop suggests that it provides a good model of the first reversible encounter complex, the so-called Michaelis-type complex, which has previously been studied kinetically (9). This complex is converted to the final complex via multiple steps, including irreversible acylation and several reversible steps (10 -12). The nature of these steps is debated. Evidence has been given that one or several conformational changes occur before acylation of the P1 amino acid (11, 13-15). These conformational changes were rate-limiting for some serpin-proteinase pairs (11, 13). However, other authors u...

“…The step EI* 3 EI may include several reversible or irreversible steps like translocation and acylation as shown previously (13). Scheme 2 predicts (30) the result shown in Equation 6.…”

Section: Kinetics Of the Inhibition Of Active Trypsins By At And P 1 mentioning

confidence: 59%

Comparative Trajectories of Active and S195A Inactive Trypsin upon Binding to Serpins

Mellet¹,

Hedstrom²,

Cahoon³

et al. 2002

“…I to E*I* via EI a , EI b , and EI c -In formation of E*I* from E and I we know, or can safely assume on the basis of prior work (7,8), that the following must occur: (a) the RCL assumes the canonical conformation in fitting into the active site of Chtr; (b) there is a major insertion of the RCL into ␤-sheet A (s4A), although how much insertion occurs is unclear; (c) the structure of Chtr within the complex changes considerably vis-à -vis the native structure, with concomitant inactivation of its catalytic apparatus (14 -16); and (d) in Chtr is acylated with ACT at residue P1 (11,12), with formation of the postcomplex fragment, a reaction that proceeds via a tetrahedral intermediate.…”

Section: Discussionmentioning

confidence: 99%

Antichymotrypsin Interaction with Chymotrypsin

Luo¹,

Zhou²,

Cooperman

1999

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9) and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10 -P14, shows facile movement in and out of ␤-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the ␣ 1 -antichymotrypsin⅐␣-chymotrypsin encounter complex, E⅐I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of ␣ 1 -antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates,

“…PAI-1 plays a role in the control of normal fibrinolysis (6) as well as a number of physiological processes that are dependent on plasminogen activation (3,7,8). PAI-1 acts as a suicide inhibitor (9, 10) that inactivates target proteinases by trapping a stabilized acyl enzyme intermediate (11)(12)(13). Significant structural changes accompanying the reaction result in efficient and essentially irreversible impairment of the proteinase mechanism (14).…”

mentioning

confidence: 99%

“…RCL insertion results in a 70-Å translocation of the enzyme molecule from the site of the initial binding (17,18). In the stable inhibitory complex, the proteinase is attached to the C terminus of the cleaved inhibitor through an ester bond with the serine of the catalytic triad (13). The deformation of the active site (19) of the proteinase trapped in the final complex prevents the completion of the normal catalytic cycle.…”

mentioning

confidence: 99%

Mechanisms of Conversion of Plasminogen Activator Inhibitor 1 from a Suicide Inhibitor to a Substrate by Monoclonal Antibodies

Komissarov

Declerck

Shore

2002

We have delineated two different reaction mechanisms of monoclonal antibodies (mAbs), MA-8H9D4 and either MA-55F4C12 or MA-33H1F7, that convert plasminogen activator inhibitor 1 (PAI-1) to a substrate for tissue (tPA)-and urokinase plasminogen activators. MA-8H9D4 almost completely (98 -99%) shifts the reaction to the substrate pathway by preventing disordering of the proteinase active site. MA-8H9D4 does not affect the rate-limiting constants (k lim ) for the insertion of the reactive center loop cleaved by tPA (3.5 s ؊1 ) but decreases k lim for urokinase plasminogen activator from 25 to 4.0 s ؊1 . MA-8H9D4 does not cause deacylation of preformed PAI-1/proteinase complexes and probably acts prior to the formation of the final inhibitory complex, interfering with displacement of the acylated serine from the proteinase active site. MA-55F4C12 and MA-33H1F7 (50 -80% substrate reaction) do not interfere with initial PAI-1/proteinase complex formation but retard the inhibitory pathway by decreasing k lim (>10-fold for tPA). Interaction of two mAbs with the same molecule of PAI-1 has been directly demonstrated for pairs MA-8H9D4/MA-55F4C12 and MA-8H9D4/MA-33H1F7 but not for MA-55F4C12/MA-33H1F7. The strong functional additivity observed for MA-8H9D4 and MA-55F4C12 demonstrates that these mAbs interact independently and affect different steps of the PAI-1 reaction mechanism.Plasminogen activator inhibitor-1 (PAI-1) 1 (1), a representative of the serpin superfamily (2-4) is the major regulator of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators in vivo (5). PAI-1 plays a role in the control of normal fibrinolysis (6) as well as a number of physiological processes that are dependent on plasminogen activation (3,7,8). PAI-1 acts as a suicide inhibitor (9, 10) that inactivates target proteinases by trapping a stabilized acyl enzyme intermediate (11-13). Significant structural changes accompanying the reaction result in efficient and essentially irreversible impairment of the proteinase mechanism (14). The target proteinase recognizes the scissile peptide bond (P1-P1Ј) in the flexible reactive center loop (RCL) exposed to the solution. Cleavage of P1-P1Ј and acylation of the proteinase trigger rapid insertion of the RCL as strand 4 of ␤-sheet A, forming the final inhibitory complex (15, 16). RCL insertion results in a 70-Å translocation of the enzyme molecule from the site of the initial binding (17,18). In the stable inhibitory complex, the proteinase is attached to the C terminus of the cleaved inhibitor through an ester bond with the serine of the catalytic triad (13). The deformation of the active site (19) of the proteinase trapped in the final complex prevents the completion of the normal catalytic cycle. This mechanism of the inhibitory reaction probably common for all serpins agrees with the molecular models of active (20 -22) and cleaved PAI-1 (22, 23) and with the known structure of a serpin/proteinase inhibitory complex (19). Deacylation and dissociation of the proteinase during or after RCL in...