Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.     

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants.

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.     

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.     

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.     

Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex

Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a salt-bridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na (+) with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the X-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na (+), we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays. The results indicate that the affinity of all three mutants for interaction with Na (+) has been impaired. The mutant with the greatest loss of affinity for Na (+) (E217A or E217Q) also exhibited a dramatic impairment ( approximately 3-4 orders of magnitude) in its activity toward both synthetic and natural substrates. Interestingly, factor Va (fVa) restored most of the catalytic defect with prothrombin, but not with the synthetic substrate. Both Glu-217 mutants exhibited a near normal affinity for fVa in the prothrombinase assay, but a markedly lower affinity for the cofactor in a direct-binding assay. These results suggest that, similar to thrombin, an ionic interaction between Glu-217 and Lys-224 stabilizes the 220-loop of fXa for binding Na (+). They further support the hypothesis that the Na (+) and fVa-binding sites of fXa are energetically linked and that a cofactor function for fVa in the prothrombinase complex involves inducing a conformational change in the 220-loop of fXa that appears to stabilize this loop in the Na (+)-bound active conformation.     

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs     

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.     

Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex

Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXa-fVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane-bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-microm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 +/- 5000 (33 200 +/- 14 100), 20 000 +/- 4500, and 30 500 +/- 1300 binding sites per vesicle with apparent K(d) values of 76 +/- 23 (71 +/- 5), 1510 +/- 430, and 223 +/- 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 +/- 370, 20 +/- 5 nm) and fX (12 630 +/- 690, 14 +/- 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 +/- 67, 23 +/- 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 +/- 0.12 and an intrinsic K(m) value of 8.0 +/- 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X

Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa; however, with the exception of a basic recognition site for the Gla domain of fX, no other interactive site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues Asn-199, Arg-200, and Lys-201 is located at a proper height on the membrane surface to interact with either the C-terminus of the Gla domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wild-type fX and its two mutants which lack either the Gla domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX, was impaired approximately 3-fold. Further kinetic analysis revealed that kcat values with both TF mutants are impaired with no change in Km. These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.     

Functionally important residues tyrosine-171 and serine-158 in sepiapterin reductase.

The active site of sepiapterin reductase (SPR), which is a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family and acts as the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor (BH4), was investigated by truncation and site-directed mutagenesis. The truncation mutants showed that N-terminal and C-terminal residues contribute to bind coenzyme and substrate, respectively. The mutant rSPRA29V showed decreased activity; however, the A-X-L-L-S sequence, which has been reported as a putative pterin binding site, was estimated to preferably work as a component in the region for binding coenzyme rather than substrate. Site-directed mutants of rSPRS158D, rSPRY171V, and rSPRK175I showed low, but significant, activity having similar Km values and kcat/Km values less than 25%, for both sepiapterin and NADPH. Both amino acids Tyr-171 and Ser-158 are located within a similar distance to the carbonyl group of the substrate in the crystal structure of mouse SPR, and the double point mutant rSPRY171V+S158D was indicated to be inactive. These results showed that Ser-158, Tyr-171, and Lys-175 contributed to the catalytic activity of SPR, and both Tyr-171 and Ser-158 are simultaneously necessary on proton transfer to the carbonyl functional groups of substrate.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation

SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel β7 and β8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.     

Residue Met(156) contributes to the labile enzyme conformation of coagulation factor VIIa

Serine protease activation is typically controlled by proteolytic cleavage of the scissile bond, resulting in spontaneous formation of the activating Ile(16)-Asp(194) salt bridge. The initiating coagulation protease factor VIIa (VIIa) differs by remaining in a zymogen-like conformation that confers the control of catalytic activity to the obligatory cofactor and receptor tissue factor (TF). This study demonstrates that the unusual hydrophobic Met(156) residue contributes to the propensity of the VIIa protease domain to remain in a zymogen-like conformation. Mutation of Met(156) to Gln, which is found in the same position of the highly homologous factor IX, had no influence on the amidolytic and proteolytic activity of TF-bound VIIa. Furthermore, the mutation did not appreciably stabilize the labile Ile(16)-Asp(194) salt bridge in the absence of cofactor. VIIa(Gln156) had increased affinity for TF, consistent with a long range conformational effect that stabilized the cofactor binding site in the VIIa protease domain. Notably, in the absence of cofactor, amidolytic and proteolytic function of VIIa(Gln156) were enhanced 3- and 9-fold, respectively, compared with wild-type VIIa. The mutation thus selectively influenced the catalytic activity of free VIIa, identifying the Met(156) residue position as a determinant for the zymogen-like properties of free VIIa.     

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.     

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.     

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.     

Delineation of the third antigenic site of lysozyme by application of a novel ''surface-simulation'' synthetic approach directly linking the conformationally adjacent residues forming the site.

We have previously shown that an antigenic site in native lysozyme resides around the disulphide bridge 30-115 and incorporates Lys-33 and Lys-116 and one or both of Tyr-20 and Tyr-23. These residues fall in an imaginary line circumscribing part of the surface of the molecule and passing through the spatially adjacent residues Tyr-20, Arg-21, Tyr-23, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. The identity of the site was confirmed by demonstrating that the synthetic peptide Tyr-Arg-Tyr-Gly-Lys-Asn-Arg-Gly-Phe-Lys (which does not exist in lysozyme but simulates a surface region of it), and an analogue in which glycine replaced Tyr-23, possessed remarkable immuno-chemical reactivity that accounted entirely for the expected reactivity of the site in native lysozyme. Tyr-23 is not part of the site, and its contribution was satisfied by a glycine spacer. The novel approach presents a powerful technique for the delineation of antigenic (and other binding) sites in native proteins and confirms that these need not always comprise residues in direct peptide linkage.     

Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.     

Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme''s active-site cleft.

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.