Amino acid residue mutations uncouple cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli contains two Gly-Gly sequences that occur at junctions between domains. A previous study (Grant, G. A., Xu, X. L., and Hu, Z. (2000) Biochemistry 39, 7316-7319) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains. Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the K(m) and k(cat). Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domains, which form the active site cleft, have a significant effect on the k(cat) of the enzyme without substantially altering the enzyme's sensitivity to serine. In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition. Interestingly, even though cooperativity of inhibition can be reduced significantly, there is little apparent effect on the cooperativity of serine binding itself. An additional mutant, G336V,G337V, also reduces the cooperativity of inhibition, but in this case serine binding also is reduced to the point at which it cannot be measured by equilibrium dialysis. The double mutant G294V,G336V demonstrates that strain imposed by mutation at one hinge can be relieved partially by mutation at the other hinge, demonstrating linkage between the two hinge regions. These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled. Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme. A comparison of these values with the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme, which will allow modeling of the dynamics of domain movement.     

The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase

D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly(336)-Gly(337), is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6 degrees in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.     

Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis

The transient kinetics of glucose binding to glucokinase (GK) was studied using stopped-flow fluorescence spectrophotometry to investigate the underlying mechanism of positive cooperativity of monomeric GK with glucose. Glucose binding to GK was shown to display biphasic kinetics that fit best to a reversible two-step mechanism. GK initially binds glucose to form a transient intermediate, namely, E* x glucose, followed by a conformational change to a catalytically competent E x glucose complex. The microscopic rate constants for each step were determined as follows: on rate k1 of 557 M(-1) s(-1) and off rate k(-1) of 8.1 s(-1) for E* x glucose formation, and forward rate k2 of 0.45 s(-1) and reverse rate k(-2) of 0.28 s(-1) for the conformational change from E* x glucose to E x glucose. These results suggest that the enzyme conformational change induced by glucose binding is a reversible, slow event that occurs outside the catalytic cycle (kcat = 38 s(-1)). This slow transition between the two enzyme conformations modulated by glucose likely forms the kinetic foundation for the allosteric regulation. Furthermore, the kinetics of the enzyme conformational change was altered in favor of E x glucose formation in D2O, accompanied by a decrease in cooperativity with glucose (Hill slope of 1.3 in D2O vs 1.7 in H2O). The deuterium solvent isotope effects confirm the role of the conformational change in the magnitude of glucose cooperativity. Similar studies were conducted with GK activating mutation Y214C at the allosteric activator site that is likely involved in the protein domain rearrangement associated with glucose binding. The mutation enhanced equilibrium glucose binding by a combination of effects on both the formation of E* x glucose and an enzyme conformational change to E x glucose. Kinetic simulation by KINSIM supports the conclusion that the kinetic cooperativity of GK arises from slow glucose-induced conformational changes in GK.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.     

Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli. D-3-phosphoglycerate dehydrogenase

The regulatory and substrate binding domains of D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) from Escherichia coli are connected by a single polypeptide strand that contains a Gly-Gly sequence approximately midway between the domains. The potential flexibility of this sequence and its strategic location between major domain structures suggests that it may function in the conformational change leading from effector binding to inhibition of the active site. Site-directed mutagenesis of this region (Gly-336-Gly-337) supports this hypothesis. When bulky side chains were substituted for the glycines at these positions, substantial changes in the ability of serine to inhibit the enzyme were seen with little effect on the activity of the enzyme. The effect of these substitutions could be alleviated by placing a new glycine residue at position 335, immediately flanking the original glycine pair. On the other hand, substituting a glycine at position 338 revealed a critical role for the side chain of Arg-338. This residue may function in stabilizing the conformation about the Gly-Gly turn, resulting in a specific orientation of the adjacent domains relative to each other. Rotation about the phi or psi bonds of either Gly-336 or Gly-337 would have a profound effect on this orientation. The data are consistent with this as a role for the Gly-Gly sequence between the regulatory and substrate binding domains of PGDH.     

Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.     

A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427-->Pro mutant human phenylalanine hydroxylase: contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding.

Recombinant human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martínez, A. (1996) Eur. J. Biochem. 242, 813-821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the two oligomeric forms when expressed in Escherichia coli. The positive cooperativity of L-Phe binding to the tetrameric form both in enzyme kinetic studies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (Vmax/[S]0.5) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with L-Phe, the dimer revealed only a 1.6-fold activation. The crystal structure has identified a five-residue flexible hinge region (Asp425-Gln429) that links the beta-strand Tbeta2 (Ile421-Leu424) and the 24 residue amphipathic alpha-helix Talpha1 (Gln428-Lys452) at the C-terminus which forms an antiparallel coiled-coil structure in the center of the tetramer [Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem. 273, 16962-16967]. The potential role of this flexible hinge in the tetramerization and the conformational transition of wt-hPAH on the cooperative binding of L-Phe was examined by site-specific mutagenesis. Substitution of Thr427 by a Pro (as in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no kinetic cooperativity of L-Phe binding, the catalytic efficiency (Vmax/[S]0.5) was decreased to about 39% of the wild-type tetramer and it was not activated by L-Phe preincubation. The dimeric forms of T427P and wt-hPAH revealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the binding isotherm for L-Phe as studied by intrinsic tryptophan fluorescence measurements. Protein stability was also reduced both for the E. coli expressed and the in vitro synthesized mutant enzyme. Collectively, these results indicate that the positive cooperativity of L-Phe binding to wt-hPAH requires a tetrameric enzyme with a C-terminal flexible hinge region (Asp425-Gln429) which has a structural role in the formation of the enzyme tetramer. Furthermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on the cooperative binding of L-Phe to tetrameric wt-hPAH. This conclusion is further supported by studies on two disease (phenylketonuria)-associated mutant forms.     

Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding

The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.     

Phosphate ion partially relieves the cooperativity of effector binding in D-3-phosphoglycerate dehydrogenase without altering the cooperativity of inhibition

The binding of L-serine to phosphoglycerate dehydrogenase from E. coli displays elements of both positive and negative cooperativity. In addition, the inhibition of enzymatic activity by L-serine is also cooperative with Hill coefficients greater than 1. However, phosphate buffer significantly reduces the cooperative effects in serine binding without affecting the cooperativity of inhibition of activity. The maximal degree of inhibition and fluorescence quenching in Tris buffer occurs when an average of two serine binding sites out of four are occupied. This value increases to three out of the four sites at maximal levels of inhibition and quenching in phosphate buffer. The increase from two to three sites appears to be due to the ability of phosphate to reduce the site to site cooperative effects and render each ligand binding site less dependent on each other. The correlation between the level of inhibition and the fractional site occupancy indicates that in Tris buffer, one serine is bound to each interface at maximal effect. In the presence of phosphate, the order of binding appears to change so that both sites at one interface fill before the first site at the opposite interface is occupied. In each case, there is a good correlation between serine binding, conformational change at the regulatory site interfaces, and inhibition of enzyme activity. The observation that phosphate does not appear to have a similar effect on the cooperativity of inhibition of enzymatic activity suggests that there are two distinct cooperative pathways at work: one path between the four serine binding sites, and one path between the serine binding sites and the active sites.     

Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition.     

Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif

Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser(336), Ser(337), Ser(342), and Ser(349) in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with beta-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous beta-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both beta-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a beta-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified beta-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that beta-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed beta-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in beta-arrestin binding to CCR5.     

Hybrid tetramers reveal elements of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when l-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by approximately 58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.     

Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.     

Conformational dynamics of subtilisin-chymotrypsin inhibitor 2 complex by coarse-grained simulations

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.     

大肠杆菌glgC基因的定点突变及功能分析

摘要: 利用PCR从Escherichia coli JM109基因组中扩增到全长为1 296 bp的glgC基因编码区, 通过PCR重组方法进行点突变, 获得氨基酸突变的3个突变体, 分别是Pro295Ser(Val121Ala, Met151Ile和Val334Asp)、Gly336 Asp单点突变和Pro295Ser/Gly336Asp(Lys109Arg), 其基因分别命名为295⁺³、336和295/ 336⁺¹。将突变和未突变的基因分别克隆到pET32-a, 构建重组质粒pET-glgC、pET-295⁺³、pET-336和pET-295/ 336+1, 在文中分别简称为a、b、c和d。转化大肠杆菌BL21(DE3), 在1 mmol/L IPTG 诱导下表达。SDS- PAGE 电泳分析显示, 在约67 kDa 处有1条明显与预期大小一致的蛋白质, 表明目的基因已得到融合表达。上述转化子的碘染和糖原含量测定结果, 第336位的Gly变成Asp后, 宿主菌的糖原含量提高; Pro295Ser/Gly336Asp(Lys109Arg)的突变导致宿主菌的糖原含量与Gly336Asp突变体相近, 表明在336突变基因的基础上增加Pro295Ser的突变没有进一步加大宿主菌中AGPase酶的反馈抑制效应的降低。已有的结果显示, Pro295Ser可以降低AGPase酶的反馈抑制效应活性, 而实验中295⁺³突变基因转入宿主菌后细胞糖原含量明显降低, 推测这个结果可能是295⁺³中的Val334Asp的突变造成, 而334位的氨基酸可能是AGPase功能域中的一个重要位点。     

The role of calcium ions in factor X activation by thrombin E192Q.

Despite considerable sequence similarities, blood coagulation serine proteases exhibit remarkable specificity with respect to which zymogen they activate. The basis for this specificity presumably involves recognition of a short sequence within the extended binding pocket of the enzyme, other interactions remote from the catalytic groove, and modulation by a definite protein cofactor. In addition, Ca2+ plays a major role in most activation processes, but, because both the enzyme and its substrate interact with Ca2+, whether Ca2+ influences the substrate, the enzyme, or both remains an open question. Thrombin is not a factor X-activating enzyme, but when Glu192, 3 residues remote from the active Ser195, is replaced with glutamine, the resultant serine protease (E192Q) becomes a bovine, but not human, factor X activator. Kinetic experiments with peptides corresponding to human and bovine factor X activating sites reveal that threonine at position P2 in human (versus a valine in bovine) accounts for the species specificity. Substitution of the threonine in P2 of the human sequence with valine allows E192Q to cleave the human peptide whereas substitution of the valine in P2 of the bovine sequence with threonine hinders E192Q catalysis. Thrombin has no high affinity Ca2+ binding sites, and E192Q proteolysis of these peptides is not altered by Ca2+. The influence of Ca2+ in E192Q-mediated factor X activation provides therefore new insights into the role of the different Ca2+ binding sites in factor X. With factor X as substrate, the addition of Ca2+ enhances Kcat 4-fold but increases Km 10-fold. When the vitamin K-dependent gamma-carboxyglutamic acid domain of factor X is removed, the Km remains high with or without Ca2+ whereas Kcat still increases upon addition of the metal ion. These results suggest that factor X undergoes two metal-dependent suggest that factor X undergoes two metal-dependent transitions that influence the presentation of the activation site to activators.     

The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.

The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.     

Conformational Dynamics of Subtilisin-Chymotrypsin Inhibitor 2 Complex by Coarse-Grained Simulations

Abstract

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.