Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Thrombin inhibitor design: X-ray and solution studies provide a novel P1 determinant

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S1 pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.     

Crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).     

Prothrombinase assembly and S1 site occupation restore the catalytic activity of FXa impaired by mutation at the sodium-binding site

Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

Thermodynamics of Na+ binding to coagulation serine proteases

The sodium binding to serine proteases triggers a conformational change in the proteins that enhances the catalytic activity of the enzymes. The interaction of the cation with the protein is mediated by the hydrogen-bonding network of water molecules that embed the Na+ site. We pointed out the crucial role of the insertion loop 186a-d and the I16-D194 ion pair in the stabilization of sodium binding pocket in thrombin. This paper contributes to better explain the molecular mechanism of sodium binding for different serine proteases leading to the identification of the structural changes necessary to engineer a functional Na+ site and regulate catalytic activity in serine proteases.     

S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?

Thrombin-like enzymes isolated from snake venoms comprise a group of serine proteinases responsible for many important coagulation disorders in the envenomed victims. Besides, these proteinases have great biotechnological interest as antithrombotic agents and as diagnostic tools. However, in spite of the recent overflow of snake venom thrombin-like enzymes (SVTLEs) on protein sequence databases, there is a lack of three-dimensional (3D) structural information on this family. Without such 3D structures available many aspects of the biological function and biochemical properties of these enzymes still remain obscure. Therefore, we have gone through a series of computational techniques, which enabled us to identify the set of residues involved in molecular recognition of inhibitors bound to the S1 subsite of snake venom thrombin-like enzymes (SVTLEs) and ultimately conclude that nonpolar (van der Waals) intermolecular interactions and ligand's hydrophobicity are the most important factors affecting binding affinities to the S1 subsite of a SVTLE isolated from the venom of Lachesis muta muta (Lmm-TLE). Consequently, we have proposed that S1 subsite lipophilicity may be used to sort binding affinities of trypsin-like enzymes to small molecules by showing that the inhibitory potency of several S1-directed compounds follows subsite lipophilicity among Lmm-TLE and other three homologous proteases. Noteworthy, in the course of our analyses we determined that thrombin's S1 subsite should, in fact, be considered less lipophilic than that of trypsin if we account for the presence of the sodium-controlled water channel communicating with the S1 subsite in the coagulant enzyme.     

High resolution crystal structures of free thrombin in the presence of K(+) reveal the molecular basis of monovalent cation selectivity and an inactive slow form

Structural biology has recently advanced our understanding of the molecular mechanisms of activation and selectivity in monovalent cation activated enzymes. Here we report a 1.9 Angstrom resolution crystal structure of free thrombin, a Na(+) selective enzyme, in the presence of KCl. There are two molecules in the asymmetric unit, one with the cation site bound to K(+) and the other with this site free. The K(+)-bound form shows key differences compared with the Na(+)-bound structure that explain the different kinetics of activation. The cation-free form, on the other hand, assumes a conformation where the monovalent cation binding site is completely disordered, the S1 pocket is inaccessible to substrate and binding to exosite I is compromised by an unprecedented >20 Angstrom shift in the position of the autolysis loop. This form, named S(*), corresponds to the inactive Na(+)-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S(*) is proposed.     

Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Na+ binding to thrombin enhances the catalytic activity toward numerous synthetic and natural substrates. The bound Na+ is located in a solvent channel 16 A away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule. Molecular modeling indicates that the G184K substitution in thrombin positions the protonated epsilon-amino group of the Lys side-chain to replace the bound Na+. Likewise, the G184R substitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189. We explored whether the G184K or G184R substitution would replace the bound Na+ and yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromised up to 200-fold as a result of impaired diffusion into the S1 site and decreased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more than that of the anticoagulant substrate protein C. These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems.     

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.     

Thrombin

Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarizes our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+ -free slow from E and the Na+ -bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+ -free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na + and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes.     

Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.     

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.     

Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.

Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.     

Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

Thrombin: a paradigm for enzymes allosterically activated by monovalent cations

Enzymes activated by monovalent cations are abundantly represented in plants and in the animal world. The mechanism, of activation involves formation of a ternary intermediate with the enzyme-substrate complex, or binding of the cation to an allosteric site in the protein. Thrombin is a Na+-activated enzyme with procoagulant, anticoagulant and signaling roles. The binding of Na+ influences allosterically thrombin function and offers a paradigm for regulatory control of protease activity and specificity. Here we review the molecular basis of thrombin allostery as recently emerged from mutagenesis and structural studies. The role of Na+ in blood coagulation and the evolution of serine proteases are also discussed.     

Oligosaccharide binding to barley alpha-amylase 1

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."     

Protease-activated receptor 4-like peptides bind to thrombin through an optimized interaction with the enzyme active site surface

Protease-activated receptor 4 (PAR4) is cleaved by thrombin at the R47-G48 peptide bond. Unlike PAR1, PAR4 does not contain a sequence readily predicted to interact with thrombin anion binding exosite-I. HPLC kinetic results on hydrolysis of PAR4 peptides (38-51 and 38-62) reveal that extending the sequence from the active site toward the exosite does not promote further binding interactions with thrombin. One-dimensional-proton line-broadening NMR indicates that the amino acids occupying the P(4)-P(1) positions of PAR4 (38-47), 44PAPR(47), come into direct contact with the thrombin surface. Less contact arises from the Leu43 at the P(5) position. Two-dimensional total correlation spectroscopy and two-dimensional transferred nuclear Overhauser effect spectroscropy studies on this complex reveal that Leu43 is flexible and can exhibit two conformational states. The binding mode observed for PAR4 peptides is similar to that of PAR1 peptides. PAR4 takes advantage of a distinctive sequence to optimize its interactions with the thrombin active site surface.