Alkyl isocyanates as active-site-specific reagents for serine protease. Identification of the active-site serine as the site of reaction

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3757-3765.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3757-3765

Author(s):

J W Chen ◽

B R Evans ◽

S H Yang ◽

H Araki ◽

Y Oshima ◽

...

Keyword(s):

Active Site ◽

Dna Cleavage ◽

Substrate Binding ◽

Point Mutations ◽

Transfer Reactions ◽

Strand Transfer ◽

Site Specific ◽

Arginine Residues ◽

Active Site Tyrosine ◽

Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

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BIS-ANS Binding To Human Thrombins

10.1055/s-0038-1652085 ◽

1981 ◽

Author(s):

G Metz ◽

L J Berliner

Keyword(s):

Active Site ◽

Unique Feature ◽

Enhanced Fluorescence ◽

Ans Binding ◽

Site Specific ◽

Catalytic Center ◽

Strong Binding

Bis-anilinonaphthalenesulfonate(bis-ANS) binds extremely strongly to the human thrombins accompanied by a much enhanced fluorescence for the dye. One unique feature of bis-ANS binding is an enhancement of TAME hydrolysis by α-thrombin to approximately 175% times the rate for native α-thrombin at pH 8.1 (0 M NaCl). Non-coagulant γ-thrombin is distinguished from highly coagulant α-thrombin by enhancing bis-ANS fluorescence ∼2 times more than that by the latter form. The very strong binding of this dye Kdiss<10-8 M, pH 6.5, 0.05 M Phosphate, 0.75 M NaCl) as welras its apparent binding loci situated near, but not obstructing, the catalytic center displays the powerfully useful properties of this probe, extremely sensitive to thrombin environment while not inhibiting its action. This is to be compared with the active site specific fluorophore inhibitor DAPA which prevents binding of other ligands to the thrombin active site.

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Requirements for Site-Specific Recombination in the Tyrosine-Family Recombinase Active Site

Biophysical Journal ◽

10.1016/j.bpj.2013.11.478 ◽

2014 ◽

Vol 106 (2) ◽

pp. 73a

Author(s):

Hsiu-Fang Fan

Keyword(s):

Active Site ◽

Site Specific ◽

Site Specific Recombination

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Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Genes to Cells ◽

10.1046/j.1365-2443.2001.00399.x ◽

2001 ◽

Vol 6 (2) ◽

pp. 87-97 ◽

Cited By ~ 53

Author(s):

Koyu Hara ◽

Mayumi Shiota ◽

Hiroshi Kido ◽

Yasushi Ohtsu ◽

Takahito Kashiwagi ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Serine Protease ◽

Active Site ◽

Virus Rna

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The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100101 ◽

2000 ◽

Vol 11 (1) ◽

pp. 1-22 ◽

Cited By ~ 85

Author(s):

Lloyd Waxman ◽

Paul L Darke

Keyword(s):

Serine Protease ◽

Active Site ◽

Serine Proteases ◽

Catalytic Triad ◽

Active Site Residues ◽

Virus Family ◽

Single Domain Protein ◽

Inhibitor Discovery ◽

Antiviral Chemotherapy ◽

Simplex Virus

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure—activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

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Iminosugars as Active-Site-Specific Chaperones for the Treatment of Lysosomal Storage Disorders

Iminosugars ◽

10.1002/9780470517437.ch10 ◽

2008 ◽

pp. 225-247 ◽

Cited By ~ 15

Author(s):

Jian-Qiang Fan

Keyword(s):

Active Site ◽

Lysosomal Storage Disorders ◽

Lysosomal Storage ◽

Site Specific ◽

Storage Disorders

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Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

Scientific Reports ◽

10.1038/s41598-019-55531-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Nis V. Nielsen ◽

Elfie Roedel ◽

Dipankar Manna ◽

Michael Etscheid ◽

Jens Preben Morth ◽

...

Keyword(s):

Enzymatic Activity ◽

Serine Protease ◽

Active Site ◽

Factor Vii ◽

Protease Domain ◽

Functional Changes ◽

E Coli ◽

Catalytic Activation ◽

Serine Protease Domain ◽

Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

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