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Sriram Krishnaswamy

Publications and source records attributed to Sriram Krishnaswamy.

11 recordsLinked to original sources
PubMed2005

Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.

Prothrombinase catalyzes thrombin formation by the ordered cleavage of two peptide bonds in prothrombin. Although these bonds are likely approximately 36 A apart, sequential cleavage of prothrombin at Arg-320 to produce meizothrombin, followed by its cleavage at Arg-271, are both accomplished by equivalent exosite interactions that tether each substrate to the enzyme and facilitate presentation of the scissile bond to the active site of the catalyst. We show that impairing the conformational transition from zymogen to active proteinase that accompanies the formation of meizothrombin has no effect on initial cleavage at Arg-320 but inhibits subsequent cleavage at Arg-271. Full thermodynamic rescue of this defective mutant was achieved by stabilizing the proteinase-like conformation of the intermediate with a reversible, active site-specific inhibitor. Irreversible stabilization of intact prothrombin in a proteinase-like state, even without prior cleavage at Arg-320, also enhanced cleavage at Arg-271. Our results indicate that the sequential presentation and cleavage of the two scissile bonds in prothrombin activation is accomplished by substrate bound either in the zymogen or proteinase conformations. The ordered cleavage of prothrombin by prothrombinase is driven by ratcheting of the substrate from the zymogen to the proteinase-like states.

Binding Sites↗
PubMed2005

Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding.

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role in physiologic coagulation.

Binding Sites↗
PubMed2005

The affinity of protein C for the thrombin.thrombomodulin complex is determined in a primary way by active site-dependent interactions.

The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C (PC). Interactions between PC and extended surfaces, likely contributed by TM within the IIa.TM complex, have been proposed to play a key role in PC activation. Initial velocities of PC activation at different concentrations of PC and TM could be accounted for by a model that did not require consideration of direct binding interactions between PC and TM. Reversible inhibitors directed toward the active site of IIa within the IIa.TM complex behaved as classic competitive inhibitors of both peptidyl substrate cleavage as well as PC activation. The ability of these small molecule inhibitors to block PC binding to the enzyme points to a principal role for active site-dependent substrate recognition in determining the affinity of IIa.TM for its protein substrate. Selective abrogation of active site docking by mutation of the P1 Arg in PC to Gln yielded an uncleavable derivative (PC(R15Q)). PC(R15Q) was a poor inhibitor (K(i) >or= 30 microm) of PC activation as well as peptidyl substrate cleavage by IIa.TM. Thus, inhibition by PC(R15Q) most likely results from its ability to weakly interfere with active site function rather than by blocking extended interactions with the enzyme complex. The data suggest a primary role for active site-dependent substrate recognition in driving the affinity of the IIa.TM complex for its protein substrate. Interactions between PC and extended surfaces contributed by IIa and/or TM within the IIa.TM complex likely contribute in a secondary or minor way to protein substrate affinity.

Antithrombins↗
PubMed2004

Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin.

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.

Amino Acid Sequence↗
PubMed2004

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study.

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.

Animals↗
PubMed2004

Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III.

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.

Animals↗
PubMed2002

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.

Culture Media, Conditioned↗
PubMed2002

Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.

Amino Acid Sequence↗
PubMed2002

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase.

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.

Animals↗
PubMed2002

The contribution of factor Xa to exosite-dependent substrate recognition by prothrombinase.

Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (alphaBFX-2b) to investigate the contributions of surfaces on the proteinase to exosite-mediated protein substrate recognition by prothrombinase. alphaBFX-2b bound reversibly to a fluorescent derivative of factor Xa (K(d) = 17.1 +/- 5.6 nm) but had no effect on active site function of factor Xa or factor Xa saturably assembled into prothrombinase. In contrast, alphaBFX-2b was a slow, tight binding inhibitor of the cleavage of either prethrombin 2 or meizothrombin des-fragment 1 by prothrombinase (K(i)(*) = 0.55 +/- 0.05 nm). Thus, alphaBFX-2b binding to factor Xa within prothrombinase selectively leads to the inhibition of protein substrate cleavage without interfering with active site function. Inhibition kinetics could adequately be accounted for by a kinetic model in which prethrombin 2 and alphaBFX-2b bind in a mutually exclusive way to prothrombinase. These are properties expected of an exosite-directed inhibitor. The site(s) on factor Xa responsible for antibody binding were evaluated by identification of immunoreactive fragments following chemical digestion of human and bovine Xa and were further confirmed with a series of recombinantly expressed fragments. These approaches suggest that residues 82-91 and 102-116 in the proteinase domain contribute to alphaBFX-2b binding. The data establish this antibody as a prototypic exosite-directed inhibitor of prothrombinase and suggest that the occlusion of a surface on factor Xa, spatially removed from the active site, is sufficient to block exosite-dependent recognition of the protein substrate by prothrombinase.

Amino Acid Sequence↗