Evidence of kinetic control of ligand binding and staged product release in MurA (enolpyruvyl UDP-GlcNAc synthase)-catalyzed reactions .

MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

Catalytic residues and an electrostatic sandwich that promote enolpyruvyl shikimate 3-phosphate synthase (AroA) catalysis.

Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.

Structural effects of a covalent linkage between antithrombin and heparin: covalent N-terminus attachment of heparin enhances the maintenance of antithrombin's activated state.

We have produced a molecule comprising of permanently-activated covalently linked antithrombin and heparin (ATH). This study was designed to elucidate the covalent linkage point(s) for heparin on antithrombin and conformational properties of the ATH molecule. ATH was produced using Schiff base/Amadori rearrangement by incubating antithrombin with unfractionated heparin for 14 d at 40 degrees C. ATH was then digested using Proteinase K, and the heparin-peptide was reacted with NaIO4/NaBH4/mild acid to degrade the heparin moiety. Sequencing of the remaining peptide was performed by Edman degradation with linkage point confirmation by LC-MS. The degree of insertion of the reactive center loop (RCL) of antithrombin into the A-sheet of ATH was examined using synthesized antithrombin RCL peptides. Binding between the peptides and ATH, and the formation of ATH in the presence of the peptides were tested. CD was used to further examine the secondary and tertiary structures of ATH. The results suggest that heparin is conjugated to the amino terminal of antithrombin in the majority of ATH molecules, proximal to the previously determined heparin binding domain of antithrombin. From the linkage data, a model is proposed for the structure of ATH. Studies using the RCL peptides and CD analysis of ATH support this model.

Biodistribution of covalent antithrombin-heparin complexes.

We have developed a covalent antithrombin-heparin (ATH) complex with advantages compared to non-covalent antithrombin:heparin (AT:H) mixtures. In addition to increased activity, ATH has a longer intravenous half-life that is partly due to reduced plasma protein binding. Given ATH's altered clearance, we investigated biodistribution of ATH in vivo. ATH made from either human plasma-derived AT (pATH) or recombinant human (produced in goats) AT (rhATH) was studied. 125I-ATH + unlabeled carrier was injected into rabbits at different doses. 131I-labeled albumin was administered just before sacrifice as a marker for trapped blood in tissues. Immediately after sacrifice, animal components were removed, weighed, and subsamples were counted for gamma-radioactivity. Percent recoveries of ATH in various organs/compartments at different time points were calculated, and kinetic distribution plots generated. At saturating doses, early disappearance of rhATH from the circulation was much faster than pATH. Co-incident with clearance, 26 +/- 3% of dose for rhATH was liver-associated compared to only 3.7 +/- 0.5% for pATH by 20 min. Also, at early time periods, >60% of all extravascular ATH was liver-associated. Analysis of the vena cava and aorta suggested that vessel wall binding might also account for initial plasma loss of rhATH. By 24 h, most of pATH and rhATH were present as urinary degradation products (51 +/- 3% and 63 +/- 8%, respectively). In summary, systemic elimination of ATH is greatly influenced by the form of AT in the complex, with liver uptake and degradation playing a major role.

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

Vimentin exposed on activated platelets and platelet microparticles localizes vitronectin and plasminogen activator inhibitor complexes on their surface.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin- or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K(d) of 80 nm) binding interaction with the NH(2) terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only -5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn.PAI-1 complexes.

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.