ChemInform Abstract: Synthesis, Evaluation, and Crystallographic Analysis of L-371,912: A Potent and Selective Active-Site Thrombin Inhibitor.

ChemInform ◽  
2010 ◽  
Vol 28 (20) ◽  
pp. no-no
Author(s):  
T. A. LYLE ◽  
Z. CHEN ◽  
S. D. APPLEBY ◽  
R. M. FREIDINGER ◽  
S. J. GARDELL ◽  
...  
Keyword(s):  
Active Site ◽  
Crystallographic Analysis ◽  
Thrombin Inhibitor

Synthesis, evaluation, and crystallographic analysis of L-371,912: A potent and selective active-site thrombin inhibitor

1997 ◽  
Vol 7 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Terry A. Lyle ◽  
Zhongguo Chen ◽  
Sandra D. Appleby ◽  
Roger M. Freidinger ◽  
Stephen J. Gardell ◽  
...  
Keyword(s):  
Active Site ◽  
Crystallographic Analysis ◽  
Thrombin Inhibitor

scholarly journals Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

2019 ◽  
Vol 20 (10) ◽  
pp. 2460
Author(s):  
Shouhei Mine ◽  
Masahiro Watanabe
Keyword(s):  
Molecular Evolution ◽  
Primary Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Thermostable Enzyme ◽  
Crystallographic Analysis ◽  
Evolutionary Pathway ◽  
Chitosan Oligosaccharides ◽  
Tim Barrel ◽  
Structural Insights

The archaeal exo-β-d-glucosaminidase (GlmA), a thermostable enzyme belonging to the glycosidase hydrolase (GH) 35 family, hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a novel enzyme in terms of its primary structure, as it is homologous to both GH35 and GH42 β-galactosidases. The catalytic mechanism of GlmA is not known. Here, we summarize the recent reports on the crystallographic analysis of GlmA. GlmA is a homodimer, with each subunit comprising three distinct domains: a catalytic TIM-barrel domain, an α/β domain, and a β1 domain. Surprisingly, the structure of GlmA presents features common to GH35 and GH42 β-galactosidases, with the domain organization resembling that of GH42 β-galactosidases and the active-site architecture resembling that of GH35 β-galactosidases. Additionally, the GlmA structure also provides critical information about its catalytic mechanism, in particular, on how the enzyme can recognize glucosamine. Finally, we postulate an evolutionary pathway based on the structure of an ancestor GlmA to extant GH35 and GH42 β-galactosidases.

CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
W E Holmes ◽  
H R Lijnen ◽  
D Collen
Keyword(s):  
Sequence Analysis ◽  
Complex Formation ◽  
Active Site ◽  
Activity Analysis ◽  
Site Directed Mutagenesis ◽  
Thrombin Inhibitor ◽  
Directed Mutagenesis ◽  
Reactive Site ◽  
Antithrombin Activity ◽  
Bovine Trypsin

α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.

Active site titration of bovine β-trypsin by Nα -(N,N -dimethylcarbamoyl)-α-aza-lysine p -nitrophenyl ester: kinetic and crystallographic analysis

FEBS Letters ◽  
1995 ◽  
Vol 358 (1) ◽  
pp. 53-56 ◽  
Author(s):  
Patrizia Sartori ◽  
Kristina Djinovic Carugo ◽  
Raffaella Ferraccioli ◽  
Gianni Balliano ◽  
Paola Milla ◽  
...  
Keyword(s):  
Active Site ◽  
Crystallographic Analysis ◽  
Nitrophenyl Ester

Structure and specificity of antibody molecules

1975 ◽  
Vol 272 (915) ◽  
pp. 43-51 ◽  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Spatial Proximity ◽  
Three Dimensional Model ◽  
Fab Fragment ◽  
Active Centre ◽  
Myeloma Protein ◽  
Close Spatial Proximity ◽  
Difference Fourier

The structure of the Fab' fragment of a human myeloma protein (IgG1 (λ) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular (3-sheets which are roughly parallel to each other and surround a tightly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.

scholarly journals Catalytic specificity of the Lactobacillus plantarum cystathionine γ-lyase presumed by the crystallographic analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasuyuki Matoba ◽  
Masafumi Noda ◽  
Tomoki Yoshida ◽  
Kosuke Oda ◽  
Yuka Ezumi ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactobacillus Plantarum ◽  
Sulfur Atom ◽  
Active Site ◽  
Crystallographic Analysis ◽  
Carboxyl Groups ◽  
Elimination Reaction ◽  
Transsulfuration Pathway ◽  
Lyase Activity ◽  
Major Reaction

Abstract The reverse transsulfuration pathway, which is composed of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL), plays a role to synthesize l-cysteine using l-serine and the sulfur atom in l-methionine. A plant-derived lactic acid bacterium Lactobacillus plantarum SN35N has been previously found to harbor the gene cluster encoding the CBS- and CGL-like enzymes. In addition, it has been demonstrated that the L. plantarum CBS can synthesize cystathionine from O-acetyl-l-serine and l-homocysteine. The aim of this study is to characterize the enzymatic functions of the L. plantarum CGL. We have found that the enzyme has the high γ-lyase activity toward cystathionine to generate l-cysteine, together with the β-lyase activity toward l-cystine to generate l-cysteine persulfide. By the crystallographic analysis of the inactive CGL K194A mutant complexed with cystathionine, we have found the residues which recognize the distal amino and carboxyl groups of cystathionine or l-cystine. The PLP-bound substrates at the active site may take either the binding pose for the γ- or β-elimination reaction, with the former being the major reaction in the case of cystathionine.

ChemInform Abstract: Structure-Activity and Crystallographic Analysis of a New Class of Non-amide-based Thrombin Inhibitor.

ChemInform ◽  
2010 ◽  
Vol 31 (18) ◽  
pp. no-no
Author(s):  
Tianbao Lu ◽  
Richard M. Soll ◽  
Carl R. Illig ◽  
Roger Bone ◽  
Larry Murphy ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Thrombin Inhibitor ◽  
New Class ◽  
Structure Activity ◽  
Abstract Structure
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