Studies of the catalytic mechanism of an active-site mutant (Y14F) of .DELTA.5-3-ketosteroid isomerase by kinetic deuterium isotope effects

Biochemistry ◽  
1991 ◽  
Vol 30 (45) ◽  
pp. 10858-10865 ◽  
Author(s):  
Liang Xue ◽  
Paul Talalay ◽  
Albert S. Mildvan
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Ketosteroid Isomerase ◽  
Deuterium Isotope Effects

Catalytic mechanism of an active-site mutant (D38N) of .DELTA.5-3-ketosteroid isomerase

Biochemistry ◽  
1991 ◽  
Vol 30 (20) ◽  
pp. 4991-4997 ◽  
Author(s):  
Liang Xue ◽  
Athan Kuliopulos ◽  
Albert S. Mildvan ◽  
Paul Talalay
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Ketosteroid Isomerase

Inhibitor Binding to the Binuclear Active Site of Tyrosinase: Temperature, pH, and Solvent Deuterium Isotope Effects

Biochemistry ◽  
1994 ◽  
Vol 33 (19) ◽  
pp. 5739-5744 ◽  
Author(s):  
Jennifer S. Conrad ◽  
Sharon R. Dawso ◽  
Esmine R. Hubbard ◽  
Theresa E. Meyers ◽  
Kenneth G. Strothkamp
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
Inhibitor Binding ◽  
Deuterium Isotope Effects

scholarly journals Free-energy profiles for catalysis by dual-specificity phosphatases

2006 ◽  
Vol 399 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Guilherme M. Arantes
Keyword(s):  
Free Energy ◽  
Active Site ◽  
Drug Targets ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Cell Signalling ◽  
Protein Tyrosine Phosphatases ◽  
Energy Profiles ◽  
Dual Specificity ◽  
Free Energy Profiles

PTPs (protein tyrosine phosphatases) are fundamental enzymes for cell signalling and have been linked to the pathogenesis of several diseases, including cancer. Hence, PTPs are potential drug targets and inhibitors have been designed as possible therapeutic agents for Type II diabetes and obesity. However, a complete understanding of the detailed catalytic mechanism in PTPs is still lacking. Free-energy profiles, obtained by computer simulations of catalysis by a dual-specificity PTP, are shown in the present study and are used to shed light on the catalytic mechanism. A highly accurate hybrid potential of quantum mechanics/molecular mechanics calibrated specifically for PTP reactions was used. Reactions of alkyl and aryl substrates, with different protonation states and PTP active-site mutations, were simulated. Calculated reaction barriers agree well with experimental rate measurements. Results show the PTP substrate reacts as a bi-anion, with an ionized nucleophile. This protonation state has been a matter of debate in the literature. The inactivity of Cys→Ser active-site mutants is also not fully understood. It is shown that mutants are inactive because the serine nucleophile is protonated. Results also clarify the interpretation of experimental data, particularly kinetic isotope effects. The simulated mechanisms presented here are better examples of the catalysis carried out by PTPs.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

Mechanism of hydration and isomerisation of 4-ethylidene-2,6-di-tert-butyl-2,5-cyclohexadien-1-one. Kinetics, acid-base catalysis and solvent deuterium isotope effects

1979 ◽  
Vol 44 (1) ◽  
pp. 110-122 ◽  
Author(s):  
Jiří Velek ◽  
Bohumír Koutek ◽  
Milan Souček
Keyword(s):  
Aqueous Medium ◽  
Rate Equation ◽  
Kinetic Data ◽  
Isotope Effects ◽  
Base Catalysis ◽  
Quinone Methide ◽  
Reaction Products ◽  
Acid Base ◽  
Deuterium Isotope Effects ◽  
Hydroxybenzyl Alcohol

Competitive hydration and isomerisation of the quinone methide I at 25 °C in an aqueous medium in the region of pH 2.4-13.0 was studied spectrophotometrically. The only reaction products in the studied range of pH are 4-hydroxybenzyl alcohol (II) and 4-hydroxystyrene (III). The form of the overall rate equation corresponds to a general acid-base catalysis. The mechanism of both reactions for three markedly separated pH regions is discussed on the basis of kinetic data and solvent deuterium effect.

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