scholarly journals Characteristics and composition of the vitamin K-dependent γ-glutamyl carboxylase-binding domain on osteocalcin

2002 ◽  
Vol 364 (1) ◽  
pp. 323-328 ◽  
Author(s):  
Roger J.T.J. HOUBEN ◽  
Dirk T.S. RIJKERS ◽  
Thomas B. STANLEY ◽  
Francine ACHER ◽  
Robert AZERAD ◽  
...  
Keyword(s):  
Binding Site ◽  
Vitamin K ◽  
Active Site ◽  
Substrate Binding ◽  
Direct Interaction ◽  
Binding Domain ◽  
High Affinity ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Gla Domain

Two different sites on vitamin K-dependent γ-glutamyl carboxylase (VKC) are involved in enzyme—substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-Km substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the γ-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.

scholarly journals A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1970 ◽  
Vol 117 (1) ◽  
pp. 169-176 ◽  
Author(s):  
W. G. Bardsley ◽  
C. M. Hill ◽  
R. W. Lobley
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Enzyme Preparation ◽  
Diamine Oxidase ◽  
Substrate Binding ◽  
Pig Kidney ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Rf Values

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with RF values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme–substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0–9.0 Å from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.

The NOACs: clinical pharmacology

ESC CardioMed ◽  
2018 ◽  
pp. 268-272
Author(s):  
Jeffrey Weitz
Keyword(s):  
Atrial Fibrillation ◽  
Vitamin K ◽  
Active Site ◽  
Randomized Clinical Trials ◽  
Vitamin K Antagonists ◽  
Oral Anticoagulants ◽  
Factor Xa ◽  
Vitamin K Antagonist ◽  
Phase Iii ◽  
High Affinity

The limitations of vitamin K antagonists prompted the development of new oral anticoagulants that could be administered in fixed doses without routine coagulation monitoring. Focusing on thrombin and factor Xa because of their prominent roles in coagulation, structure-based design led to the development of small molecules that bind to the active site pockets of these enzymes with high affinity and specificity. Four non-vitamin K antagonist oral anticoagulants are now licensed: dabigatran, which inhibits thrombin, and rivaroxaban, apixaban, and edoxaban, which inhibit factor Xa. In phase III randomized clinical trials that included over 100,000 patients these agents have proven to be at least as effective as vitamin K antagonists for prevention of stroke in patients with non-valvular atrial fibrillation and for treatment of venous thromboembolism, and to produce less bleeding, particularly less intracranial bleeding.

scholarly journals Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of theBacillus subtilisandEscherichia coliSecA Protein

1995 ◽  
Vol 270 (32) ◽  
pp. 18975-18982 ◽  
Author(s):  
Jeroen P. W. van der Wolk ◽  
Michael Klose ◽  
Janny G. de Wit ◽  
Tanneke den Blaauwen ◽  
Roland Freudl ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Domain ◽  
Atp Binding ◽  
Atp Binding Site ◽  
High Affinity ◽  
Magnesium Binding

Steps in the reactions of proteolytic enzymes with their substrates

1970 ◽  
Vol 257 (813) ◽  
pp. 105-110 ◽  
Keyword(s):  
Active Sites ◽  
Dissociation Constants ◽  
Proteolytic Enzymes ◽  
Substrate Binding ◽  
Hydrolytic Enzymes ◽  
Dimensional Structure ◽  
Chemical Information ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Wide Range

Kinetic experiments should be designed to answer specific questions about a reaction mechanism. The present paper is intended to show how a number of specific questions have been answered. Chymotrypsin and trypsin are mainly used to illustrate the different approaches, but many of the arguments used are equally applicable to the reactions of other hydrolytic enzymes with serine-OH or cysteine-SH at the active site. T he recognition of serine-OH and cysteine-SH as essential groups at the active sites of different hydrolytic enzymes did not rest on kinetic evidence. This was deduced from the correlation of enzyme activity with the extent of modification of specially reactive groups. The investigation of proton dissociation equilibria and the assignment of dissociation constants to groups with specified functions in substrate binding, catalysis or protein conformation was the first objective of serious kinetic studies of enzyme reactions. Steady state rate measurements over a wide range of pH showed that groups with p K 6.25 and 6.85 respectively are involved in the catalytic activity of trypsin and chymotrypsin with certain specific substrates (Hammond & Gutfreund 1955). In the case of chymotrypsin it was also shown by Hammond & Gutfreund (1955) that a group with a more alkaline pK is involved in substrate binding. This latter group was subsequently identified and its function was elucidated through the elegant experiments of Oppenheimer, Labouresse & Hess (1966). The identification of histidine as the group with p K A near neutrality, involved in the catalytic mechanism of trypsin and chymotrypsin, was subsequently confirmed by direct chemical methods by Schoelmann & Shaw (1963). Only kinetic analysis can demonstrate the involvement of proton donors or acceptors with specific properties in enzyme-substrate interaction or in catalysis. The clear identification of chemical groups capable of performing such functions is coming from the crystallographic analysis of the three-dimensional structure at the site of enzyme-substrate interaction, as illustrated in other papers presented in this discussion. Very interesting chemical information is obtained when the effect of structure on reactivity is synthesized from the composite of crystallographic and kinetic data.

Zinc Binding to Protein C and Activated Protein C Modulates Their Interaction with Endothelial Cell Protein C Receptor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 331-331
Author(s):  
Prosenjit Sen ◽  
Sanghamitra Sahoo ◽  
Usha Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Catalytic Activity ◽  
Vitamin K ◽  
Binding Sites ◽  
Protein C ◽  
Zinc Binding ◽  
Zinc Ions ◽  
Clotting Factors ◽  
Binding Studies ◽  
High Affinity ◽  
Gla Domain

Abstract Abstract 331 Introduction/background: Zinc is a multi-functional element that is essential for life and the second most abundant metal ion, after iron in eukaryotic organisms. Zinc deficiency has been associated with bleeding disorders and with defective platelet aggregation, suggesting it may play an important role in hemostasis. Zinc ions have been shown to enhance activation of the intrinsic pathway of coagulation but to down-regulate the extrinsic pathway of coagulation. All vitamin K-dependent coagulation proteins have calcium binding sites and may therefore to some extent, interact with other divalent metal ions, including zinc, through these sites. Recent crystallography studies identified a pair of Zn2+ binding sites in FVIIa protease domain, and with the exception of Glu220, all the side chains involved in both the Zn1 and Zn2 coordination in FVIIa are unique to FVIIa and are not present in other vitamin K-dependent clotting factors (Bajaj et al., J Biol Chem 2006; 281:24873-88). Nonetheless, Zn2+ may bind to other vitamin K-dependent clotting factors at sites different from those identified in FVIIa. Objective: The aim of the present study is to investigate the effect of zinc ions on the protein C pathway, particularly on protein C/APC binding to EPCR, protein C activation and APC catalytic activity. Methods: Protein C and APC binding to EPCR on endothelial cells was examined by radioligand binding studies. Protein C activation and APC catalytic activity were evaluated in chromogenic assays. Equilibrium dialysis was used to measure zinc binding to protein C/APC. Conformational changes in protein C/APC were monitored by intrinsic fluorescence quenching. Results: Zn2+ does not replace the Ca2+ as a mandatory cofactor for protein C/APC binding to EPCR but Zn2+ at physiologically relevant concentrations (10 to 25 μM) markedly increased Ca2+-dependent protein C and APC binding to EPCR (∼2 to 5-fold). The kinetic analysis of protein C and APC binding to EPCR suggested that Zn2+ enhanced protein C/APC binding to EPCR by increasing the binding affinity of protein C/APC to its receptor (Kd for APC: – Zn2+, 117 ± 27 nM; + Zn2+, 9.3 ± 3.3 nM; Kd, for protein C: – Zn2+, 96 ± 26 nM; + Zn2+, 21.4 ± 6.6 nM). The enhancing effect of Zn2+ on APC binding to EPCR was also observed in the presence of physiological concentrations of Mg2+, which itself increased the APC binding to EPCR, two-fold. Zn2+-mediated increased protein C binding to EPCR resulted in increased APC generation. The effect of Zn2+ was not limited to enhancing protein C and APC binding to EPCR but also affected the catalytic activity of APC. Zn2+ inhibited the amidolytic activity of APC half-maximally at 50 to 100 μM. Zn2+ also inhibited the amidolytic activity of Gla domain deleted (GD)-APC in a similar fashion. The inhibitory effect of Zn2+ was partially reversed by physiological concentrations of calcium. Addition of Zn2+ to protein C or APC quenched the intrinsic fluorescence of both APC and GD-APC. Data from the equilibrium binding studies performed with 65Zn2+ revealed that Zn2+ binds to both GD-APC and APC, but that the amount of Zn2+ bound to APC was 3 to 4-fold higher than the amount bound to GD-APC. Kinetic analysis of equilibrium binding studies suggested that two Zn2+ atoms bind to APC outside the Gla domain with relatively high affinity (∼70 μM). At least one of the Zn2+ sites may overlap with the Ca2+ binding site as the Zn2+ binding to GD-APC was inhibited by approximately 50% by saturating concentrations of Ca2+. The substantially increased Zn2+ binding to the APC compared to GD-APC suggested that the N-terminus of the Gla domain of protein C contains multiple Zn2+ binding sites. Interestingly, Zn2+ bound to APC and GD-APC with a similar high affinity suggesting that the Gla domain, as well as the protease domain, may contain high affinity binding sites for Zn2+. A majority of the Zn2+ binding sites in the Gla domain appear to be distinct from the Ca2+ binding sites as less than 40% of the maximal Zn2+ binding could be blocked by Ca2+. The putative zinc binding sites in protein C/APC appeared to be unique as no consensus canonical zinc binding sequences homologous to other known zinc binding proteins were found in protein C. Conclusions: Our present data show that Zn2+ binds to protein C/APC and induces a conformational change in these proteins, which in turn leads to higher affinity binding to their cellular receptor EPCR. Overall our results suggest that zinc ions may play an important regulatory role in the protein C pathway. Disclosures: No relevant conflicts of interest to declare.

Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 30-30 ◽  
Author(s):  
Raffaella Toso ◽  
Hua Zhu ◽  
Rodney M. Camire
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Membrane Surface ◽  
Coagulation Factor ◽  
Salt Bridge ◽  
Kinetic Studies ◽  
Factor X ◽  
Bridge Formation ◽  
High Affinity ◽  
Km Value

Abstract The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.

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