Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Henriksen, Ruth Ann; Mann, Kenneth G.

doi:10.1021/bi00431a017

Cited by 38 publications

(30 citation statements)

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“…The S 1 pockets of THRO and TRYP are similar, except for a Ser190Ala substitution, because of which the pocket is enlarged and a hydrogen‐bonding partner for the P 1 residue is lost [72]. In agreement with TRYP mutagenesis results (G216A, G226A variants [74]), the G226V mutation introduced into THRO results in a loss of activity (probably due to the disturbance of Asp189 orientation) as observed for Quick II, the naturally occurring THRO mutant [75]. This is, however, less deleterious for Arg, as it interacts directly with Asp189 and fills the greater space of the pocket better.…”

Section: Specialized Tryp‐like Proteasesmentioning

confidence: 72%

Structural and energetic determinants of the S₁‐site specificity in serine proteases

Czapinska

Otlewski

1999

European Journal of Biochemistry

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In recent years the number of determined three-dimensional structures of serine proteases that are accompanied by detailed mutational studies has grown rapidly. In particular, spatial structures have been described for enzymes involved in processes of critical medical significance, often related to severe pathophysiological diseases. There has also been significant progress in the understanding of the structural grounds for the substrate specificity of serine proteases. This review is concerned mainly with primary structural determinants of the S 1 specificity, the crucial component of substrate selectivity, often in relation to more distant specificity elements, which cooperatively influence the S 1 site.Keywords: enzyme specificity; protein inhibitors; proteolytic enzymes; serine proteases; specificity pocket.Serine proteases carry out an unlimited number of hydrolytic reactions making use of an extremely broad range of substrate specificities [1]. They have been studied intensively over the past four decades, leading to the accumulation of high-quality data in terms of amino acid and nucleotide sequences, threedimensional structures, and kinetic and thermodynamic constants for their substrates and inhibitors. Despite the long investigation into this family, the results of recent research in the area of serine proteases are spectacular. Three new, structurally different, families have been found: the wheat serine carboxypeptidase II family (Fig. 1C) [2], the human cytomegalovirus serine proteinase family with the His/His/Ser catalytic triad [3], and the proteolytic component ClpP of the ATP-dependent protease family [4]. Thus, together with the chymotrypsin (CHYM) and subtilisin families there are five known distinct folds of serine proteases. Moreover, human rhinovirus 3C protease, despite a very low sequence homology and a Glu/His/Cys catalytic triad, folds in a manner similar to CHYM-like enzymes [5]. The catalytic subunit of proteasome is a novel type threonine protease, which can also function as a serine protease, i.e. after the substitution of catalytic Thr by Ser [6]. Interestingly, the recently determined structure of a typical trypsin-like enzyme, human tryptase (TRYPT) is reminiscent of proteasome, the enzyme is active as a heparin-stabilized tetramer with four active sites directed towards a central pore [7]. Moreover, other protein folds can serve as serine proteases, for example, antibodies after immunization with transition-state analogs [8] and prolyl isomerase with an engineered high amidase activity toward P 1 Pro-containing substrates [9]. This review focuses on recent achievements in serine proteases from the CHYM and subtilisin families, and in particular on the recently determined three-dimensional structures of free enzymes and their inhibitor complexes. Enzymes taking part in blood clotting and fibrinolysis, and other proteases of medical importance are emphasized in particular. Much emphasis is also put on the results of mutagenic studies intended to narrow, broaden or change protease ...

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Section: Specialized Tryp‐like Proteasesmentioning

confidence: 72%

Structural and energetic determinants of the S₁‐site specificity in serine proteases

Czapinska

Otlewski

1999

European Journal of Biochemistry

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“…63.2 s and 67.2 s, respectively, for approximately 100 ng of ecarin‐activated thrombin. In prothrombin mutations where the primary defect was determined to be the inability to cleave fibrinogen, such as Quick II [10] or Perijá[12], there was no detectable clotting activity. However, this simple assay does not rule out subtle effects on thrombin–fibrinogen interactions, neither has the mutant thrombin been tested on other substrates.…”

Section: Resultsmentioning

confidence: 99%

A common mutation, Arg457→Gln, links prothrombin deficiencies in the Puerto Rican population

Lefkowitz¹,

Weller²,

Nuss

et al. 2003

Journal of Thrombosis and Haemostasis

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Summary. Five unrelated families with Puerto Rican ancestry were identi®ed as having at least one member with bleeding due to a prothrombin de®ciency. Genetic prothrombin de®ciencies are extremely rare, but at the University of Puerto Rico Hemophilia Center, prothrombin de®ciency is the third most common congenital coagulation factor de®ciency. Because Puerto Rico is relatively isolated, there was a reasonable expectation of a founder effect. Prothrombin genes from probands and their parents were directly sequenced from PCR ampli®ed exons using forward and reverse primers. Four novel prothrombin mutations were identi®ed. The ®rst, a G3A substitution at DNA position 10150 predicting an Arg4573Gln (R457Q) replacement, is common to all ®ve families. In two of the families, the proband children are homozygous for R457Q. In the other three families, the probands are compound heterozygotes for R457Q and one of the other three mutations, which include another point mutation (g16Q), a deletion and a splice junction mutation. The two point mutations have been designated Puerto Rico I and Puerto Rico II. The crystal structure of a-thrombin predicts that the R457Q mutation removes a salt bridge that links the A-and B-chains of thrombin. The primary effect of this defect appears to be destabilization of the circulating prothrombin, creating a moderate hypoprothrombinemia. However, prothrombin antigen/activity ratios indicate a dysprothrombinemia as well, most likely due to the inability of R457Q prothrombin to activate fully to thrombin.

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“…It may be the nature of the amino acid change at position 418 that leads to the decreased enzyme efficacy observed in prothrombin Tokushima. A Gly -+ Val substitution at position 558 is found in prothrombin Quick 11 (42). Gly-558 is adjacent to the Cys-551 to Tyr-557 loop segment (20) and is conserved in all 11 species.…”

Section: -----F-vndt-------f-vndt--------s-eds--------s-eds__pnb -F--mentioning

confidence: 99%

“…Gly-558 is adjacent to the Cys-551 to Tyr-557 loop segment (20) and is conserved in all 11 species. Substitution of Val for Gly appears to alter the primary substrate-binding pocket (42).…”

Section: -----F-vndt-------f-vndt--------s-eds--------s-eds__pnb -F--mentioning

confidence: 99%

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

Banfield

MacGillivray

1992

Proc. Natl. Acad. Sci. U.S.A.

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The cDNA sequence of the B chain of thrombin (EC 3.4.21.5) has been determined from nine vertebrate species (rat, mouse, rabbit, chicken, gecko, newt, rainbow trout, sturgeon, and hagfish). The amino acid sequence identities vary from 96.5% (rat vs. mouse) to 62.6% (newt vs. hagfish). Of the 240 amino acids spanned in all the species compared, there is identity at 110 (45.8%) positions. When conservative changes are included, the amino acid similarity increases to 75%. The most conserved portions of the B chain are the active-site residues and adjacent amino acids, the B loop, and the primary substrate-binding region. In addition, the Arg-Gly-Asp motif is conserved in 9 of the 11 species compared, and the chemotactic/growth factor domain is well conserved in all ofthe 11 species compared. The least conserved regions of the B chain correspond to surface loops, including the putative thrombomodulin-binding sites and one of the hirudin-binding regions. The extent of the amino acid sequence smilarity and the conservation of many of the functional/structural motifs suggests that, in addition to their role in blood coagulation, vertebrate thrombins may also play an important role in the general mechanisms of wound repair.The final reaction of the coagulation pathway is the conversion of fibrinogen to fibrin by the serine protease thrombin (EC 3.4.21.5) (1, 2). In mammals, thrombin is generated from its zymogen, prothrombin, by the limited proteolytic action of factor Xa in the presence of factor Va, calcium ions, and phospholipid (2). In addition, thrombin also activates/ inactivates other coagulation factors such as factor XIII, factors V and VIII, and protein C. The anticoagulant activity of thrombin is regulated through the interaction of thrombin with thrombomodulin (2, 3), an endothelial cell membrane protein related in structure to the low density lipoprotein receptor (4). While thrombomodulin likely binds to thrombin by an exposed surface loop, the precise binding site has yet to be resolved (5, 6). The thrombin/thrombomodulin complex activates protein C, which in the presence of protein S then degrades factors Va and VIlla (2).The enzymatic activity of thrombin is regulated by the endogenous protease inhibitor antithrombin III (2). Thrombin is also inhibited by protein inhibitors from nonplasma sources such as hirudin, from the European medicinal leech (7). The substrate specificity of thrombin is similar to that of trypsin, cleaving after basic amino acid residues (8). However, thrombin has a more restricted substrate range than trypsin. The molecular basis of this restricted substrate specificity is still unresolved but is thought to be the result of interactions of the substrates with secondary binding sites distant from the active site (9, 10).Thrombin is also a potent stimulator of tissue plasminogen activator release from endothelial cells (11), and various forms of thrombin are chemotactic for monocytes and neutrophils (12)(13)(14). This chemotactic activity is blocked by binding with hirudin...

show abstract

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Cited by 38 publications

References 27 publications

Structural and energetic determinants of the S₁‐site specificity in serine proteases

Structural and energetic determinants of the S₁‐site specificity in serine proteases

A common mutation, Arg457→Gln, links prothrombin deficiencies in the Puerto Rican population

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

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Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Cited by 38 publications

References 27 publications

Structural and energetic determinants of the S1‐site specificity in serine proteases

Structural and energetic determinants of the S1‐site specificity in serine proteases

A common mutation, Arg457→Gln, links prothrombin deficiencies in the Puerto Rican population

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

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Structural and energetic determinants of the S₁‐site specificity in serine proteases

Structural and energetic determinants of the S₁‐site specificity in serine proteases