Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen-like property

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those of the wild-type VIIa. Susceptibility of the alpha-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.     

Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

Thermodynamic linkage between the S1 site,the Na+ site,and the Ca2+ site in the protease domain of human activated protein C (APC). Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops

The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K(d) values of approximately 24 mm in the absence and approximately 6 mm in the presence of Ca2+. Sodium-occupied APC has approximately 100-fold increased catalytic efficiency ( approximately 4-fold decrease in K(m) and approximately 25-fold increase in k(cat)) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly ( approximately 1.2-fold). Ca2+ binds to the protease domain of APC with K(d) values of approximately 438 microm in the absence and approximately 105 microm in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K(m) but increases the k(cat) approximately 10-fold, and Na+ further increases this k(cat) approximately 3-fold and decreases the K(m) value approximately 3.7-fold. In agreement with the K(m) data, sodium-occupied APC has approximately 4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation

In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a(2) dimer by cleaving the Factor XIII activation peptide segment at the Arg(37)-Gly(38) peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K(m), k(cat), and k(cat)/K(m) were determined for thrombin hydrolysis of fibrinogen Aalpha-(7-20), Factor XIII activation peptide-(28-41), and Factor XIII activation peptide-(28-41) with a Val(34) to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28-41) exhibits a 10-fold lower k(cat)/K(m) value than fibrinogen Aalpha-(7-20). With the Factor XIII V34L mutation, decreases in K(m) and increases in k(cat) produce a 6-fold increase in k(cat)/K(m) relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.     

Deletion of the membrane anchoring region of tissue factor abolishes autoactivation of factor VII but not cofactor function. Analysis of a mutant with a selective deficiency in activity.

The activation of human blood coagulation factor VII can occur by the feedback activity of either factor VIIa (autoactivation) or factor Xa. Both of these reactions are known to be enhanced by the presence of tissue factor, an integral membrane protein and the cofactor for factor VIIa. We examine here the activation of 125I-factor VII by both factor VIIa and factor Xa employing a mutant soluble form of tissue factor which has had its transmembrane and cytoplasmic domains deleted (sTF1-219). This mutant soluble tissue factor retains cofactor activity toward factor VIIa in a single-stage clotting assay but shows a strong dependence on initial plasma levels of factor VIIa (from 1 to 10,000 ng/ml) when compared to wild-type tissue factor. We show that this dependence is due to a deficiency of sTF1-219 in ability to both promote autoactivation and enhance the factor Xa-catalyzed activation of 125I-factor VII. sTF1-219 does not, however, inhibit the tissue factor-independent activation of 125I-factor VII by factor Xa. The results strongly suggest that the phospholipid anchoring region of tissue factor is essential for autoactivation and beneficial for factor Xa-catalyzed activation of 125I-factor VII. In addition, when taken together with the dependence of clotting times on initial factor VIIa levels observed with sTF1-219, these results indicate that factor VII autoactivation may be of greater importance in the initiation of blood coagulation via tissue factor than has been previously realized.     

Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley-Liss, Inc.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro⁷⁹-Pro⁹² loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu⁸⁴ resides in the Pro⁷⁹-Pro⁹² loop, and Thr¹²¹ resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu⁸⁴ nor Thr¹²¹ makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro⁷⁹-Pro⁹² loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro⁷⁹-Pro⁹² loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.     

Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Calpha atoms of VIIa or sTF derived from NMA in the VIIa-sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Calpha atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa-sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.     

Engineered recombinant factor VII Q217 variants with altered inhibitor specificities.

Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100%, VIIQ217A 15%, and VIIQ217G <1% clotting activities relative to wild-type VII. Binding of VIIQ217A and VIIQ217G to TF was comparable to that of wild-type VII to TF. All the variants were readily activated by factor Xa. Autoactivation in the presence of TF was efficient with VIIQ217E, slow with VIIQ217A, but undetected with VIIQ217G. Relative to wild-type VII added at the same concentration, VIIQ217E had no effect on the PT of normal plasma, whereas VIIQ217A slightly and VIIQ217G dramatically prolonged the clotting time in a dose-dependent manner. Activation of macromolecular substrates paralleled this functional inhibition. The k(cat)/K(M) values for factor X activation in the presence of TF were 2.4 for VIIaQ217E as compared to 1.9 (M(-)(1) s(-)(1) x 10(7)) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x10(3) M(-)(1) min(-)(1)), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q(217) is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.     

Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k(cat)/K(m) values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K(m) values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k(cat)/K(m)) of acylated AMP hydrolysis, but not maximal catalytic turnover (k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.     

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.     

Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.     

Contribution of W229 to the transglycosylation activity of 4-alpha-glucanotransferase from Pyrococcus furiosus

A W229H mutant of 4-alpha-glucanotransferase (4-alpha-GTase) from Pyrococcus furiosus was constructed and its catalytic properties were studied to investigate the role of W229 in the catalytic specificities of the enzyme. Various activities and kinetic parameters were determined for the wild-type and W229H mutant enzymes. The transglycosylation factor and transglycosylation activity of the mutant enzyme markedly decreased, but its hydrolysis activity was scarcely affected. It was discovered that the k(cat)/K(m) value of transglycosylation activity significantly decreased to about 15% of that of the wild type, while k(cat)/K(m) value of hydrolysis activity changed little for the mutant enzyme. The hydrophobicity of W229 was thought to be critical to the transglycosylation activity of the enzyme based on the enzyme's modeled tertiary structures.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Contribution of an imidazole-indole stack to high catalytic potency of a lysine-specific serine protease, Achromobacter protease I

Achromobacter protease I (API), a lysine-specific serine-protease of the trypsin family, has an aromatic-ring stacking Trp 169-His 210 in close proximity to the reactive site. In order to investigate the role of this novel aromatic stacking, several mutants of the two residues were constructed and their kinetic parameters were determined. Three His 210 mutants showed lower activity by one order of magnitude than the wild-type with a peptide substrate of Ala-Ala-Lys-MCA (4-methylcoumaryl-7-amide), but 30-170% activity towards Val-Leu-Lys-MCA, suggesting that His 210 plays a role in keeping high activity toward various substrates by maintaining the active form of the substrate-binding subsite. Kinetic results of eight Trp 169 variants showed a roughly linear relation between k(cat) or K(m) values and the surface area at residue 169. With increasing size of the side-chain, k(cat) values increased, while K(m) values decreased. A systematic kinetic analysis of the activities of Trp 169 mutants toward Lys-MCA, Ala-Lys-MCA, and Ala-Ala-Lys-MCA peptide substrates revealed that large side-chain, rather than aromaticity, plays an important role in retaining the high catalytic activity of API. Due to the presence of the aromatic stacking, API shows one order of magnitude higher activity than bovine trypsin.