Glu192–&gt;Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor

Guinto, Enriqueta R.; Jia, Yali; Bonniec, B. F. Le; Esmon, Charles T.

doi:10.1016/s0021-9258(17)32321-9

Cited by 43 publications

(28 citation statements)

References 27 publications

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“…The alignment of the thrombin sequence with that of chymotrypsin reveals an 8 amino acid insertion between residues 60 and 61. Part of this 60-loop insertion, namely, the Tyr 60a -Pro 60b -Pro 60c -Trp 60d motif, is implicated in the P 2 and P 3 specificities of thrombin (Bode et al, 1992;Le Bonniec et al, 1993;Guinto et al, 1994), whereas its C-terminal portion extends over the presumed S′ region of the catalytic groove (Figure 1). The sixth amino acid of this loop, Lys 60f , could conceivably interact with one or more of the P 1 ′, P 2 ′, and P 3 ′ residues of a substrate.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

et al. 1996

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The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.

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Section: Resultsmentioning

confidence: 99%

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

et al. 1996

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“…The possibility that the 60 loop is more mobile than previously hypothesized was suggested by the observation that isosteric substitution of Glu192 in thrombin with Gln, thrombin E192Q, results in a mutant which binds BPTI tightly (Guinto et al, 1994;Rezaie & Esmon, 1996). For BPTI to interact with thrombin E192Q in a canonical fashion, the loop would have to move, and hence significant flexibility in the loop was proposed (Guinto et al, 1994). Less likely alternatives were that inhibition might involve some major alteration in BPTI conformation or the mechanism of BPTIthrombin inhibition.…”

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confidence: 99%

Thrombomodulin Increases the Rate of Thrombin Inhibition by BPTI

Rezaie

Esmon

1998

Biochemistry

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Thrombin undergoes allosteric modulation by thrombomodulin (TM) that results in a shift in macromolecular specificity, blocking fibrinogen clotting while enhancing protein C activation. The TM enhancement of protein C activation involves both an 8-fold decrease in Km and a 200-fold increase in kcat. Although TM-mediated conformational changes in thrombin have been detected by many techniques, the nature of these changes remains obscure. Access to the active center of thrombin is relatively restricted due to the presence of a large insertion loop at residue 60 (chymotrypsin numbering) that has been implicated in modeling studies as being responsible for poor inhibition by BPTI. Thrombin and the E192Q mutant, which binds BPTI much more tightly than thrombin, are both inhibited very slowly by BPTI. TM increases the rate of thrombin or thrombin E192Q inhibition by BPTI approximately 10-fold. When analyzed as slow tight binding inhibition, the TM effect on thrombin E192Q inhibition by BPTI is primarily on the first, reversible step in the reaction. Structural studies of the thrombin E192Q-BPTI complex have previously shown that the 60 loop lies over the BPTI, a position which requires 8 A movement at the apex of the 60 loop, and that BPTI is found in the same canonical orientation as in the trypsin complex. It follows that TM enhancement of the initial interaction of thrombin results in a conformation that favors interactions with BPTI, probably involving motion of the 60 loop.

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“…One distinctive difference in the active-site composition of the enzymes is the presence of Glu 192 in thrombin and PC, which is Gln 192 in FXa and FVIIa [106]. This difference in the negative electrostatic medium at the active site is the origin of discrimination between natural inhibitors and chromogenic substrates; Glu 192 confers resistance to Kunitz type inhibitors [102] whereas FXa is prone to inhibition by them.…”

Section: Basic Characteristics Of the Enzymes Of Bold Clotting Supporting The Catalytic Functionmentioning

confidence: 99%

“…This difference in the negative electrostatic medium at the active site is the origin of discrimination between natural inhibitors and chromogenic substrates; Glu 192 confers resistance to Kunitz type inhibitors [102] whereas FXa is prone to inhibition by them. Such phenomena were studied with the Q192E mutant of FXa [106][107][108] and the E192Q mutant of thrombin [88,105]. The E192Q mutation in thrombin resulted in higher cleavage rates of substrates that have an acidic residue at P 3 .…”

Section: Basic Characteristics Of the Enzymes Of Bold Clotting Supporting The Catalytic Functionmentioning

confidence: 99%

“…Our interest was piqued by the great importance of the absence of negative charge at 192 in FXa in the process of prothrombin activation as most of the P 3 and P 3 ' sites in human and bovine prothrombin are acidic. An important study of the activation of prothrombin by FXa and its Q192E mutant in the presence and absence of FVa indicated that FVa can compensate for the electrostatic repulsion between the Q192E mutant and the acidic P 3 and P 3 ' sites [106].…”

Section: Basic Characteristics Of the Enzymes Of Bold Clotting Supporting The Catalytic Functionmentioning

confidence: 99%

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Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System

Kovach

2021

Life

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Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C–catalyzed reactions revealed that specific binding of effectors at Sn, n = 1–4 and S’n, n = 1–3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (1H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin. The emergence of short hydrogen bonds at the binding interface of effectors and thrombin at remote exosites has recently gained recognition. Herein, I describe our contribution, a confirmation of this discovery, by low-field 1H NMR. The principal conclusion of this review is that proton sharing at distances below the sum of van der Waals radii of the hydrogen and both donor and acceptor atoms contribute to the remarkable catalytic prowess of serine proteases of the blood clotting system and other enzymes that employ acid-base catalysis. Proton bridges also play a role in tight binding in proteins and at exosites, i.e., allosteric sites, of enzymes.

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Glu192–>Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor

Cited by 43 publications

References 27 publications

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Thrombomodulin Increases the Rate of Thrombin Inhibition by BPTI

Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System

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Glu192–>Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor

Cited by 43 publications

References 27 publications

Characterization of the P2‘ and P3‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis,

Characterization of the P2‘ and P3‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis,

Thrombomodulin Increases the Rate of Thrombin Inhibition by BPTI

Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System

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Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,