Second-sphere electrostatic effects in the active site of glutathione S-transferase. Observation of an on-face hydrogen bond between the side chain of threonine 13 and the .pi.-cloud of tyrosine 6 and its influence on catalysis

1993 ◽  
Vol 115 (17) ◽  
pp. 7910-7911 ◽  
Author(s):  
Suxing Liu ◽  
Xinhua Ji ◽  
Gary L. Gilliland ◽  
Walter J. Stevens ◽  
Richard N. Armstrong
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Side Chain ◽  
Glutathione S Transferase ◽  
Electrostatic Effects

Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of β-d-glucose

2000 ◽  
Vol 347 (2) ◽  
pp. 553-559 ◽  
Author(s):  
Susanne WITT ◽  
Gerd WOHLFAHRT ◽  
Dietmar SCHOMBURG ◽  
Hans-Jürgen HECHT ◽  
Henryk M. KALISZ
Keyword(s):  
Hydrogen Bond ◽  
Glucose Oxidase ◽  
Active Site ◽  
Glucose Oxidation ◽  
Kinetic Studies ◽  
Fold Increase ◽  
Side Chain ◽  
Maximal Velocity ◽  
Active Site Residues ◽  
Hydrogen Bond Interaction

The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of β-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of β-D-glucose, to be absolutely essential for the efficient binding of β-D-glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of β-D-glucose, exhibits an 80-fold higher apparent Km (513 mM) but a Vmax only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of β-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent Km for glucose (to 733 mM) and a decrease in the Vmax to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent Km more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of β-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation.

scholarly journals Molecular Basis for the Reduced Catalytic Activity of the Naturally Occurring T560M Mutant of Human 12/15-Lipoxygenase That Has Been Implicated in Coronary Artery Disease

2011 ◽  
Vol 286 (27) ◽  
pp. 23920-23927 ◽  
Author(s):  
Kathrin Schurmann ◽  
Monika Anton ◽  
Igor Ivanov ◽  
Constanze Richter ◽  
Hartmut Kuhn ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Bond ◽  
Catalytic Activity ◽  
Active Site ◽  
Side Chain ◽  
Lag Phase ◽  
Hydrogen Bond Network ◽  
Binding Partner ◽  
Naturally Occurring ◽  
Bond Network

Lipoxygenases have been implicated in cardiovascular disease. A rare single-nucleotide polymorphism causing T560M exchange has recently been described, and this mutation leads to a near null variant of the enzyme encoded for by the ALOX15 gene. When we inspected the three-dimensional structure of the rabbit ortholog, we localized Thr-560 outside the active site and identified a hydrogen bridge between its side chain and Gln-294. This interaction is part of a complex hydrogen bond network that appears to be conserved in other mammalian lipoxygenases. Gln-294 and Asn-287 are key amino acids in this network, and we hypothesized that disturbance of this hydrogen bond system causes the low activity of the T560M mutant. To test this hypothesis, we first mutated Thr-560 to amino acids not capable of forming side chain hydrogen bridges (T560M and T560A) and obtained enzyme variants with strongly reduced catalytic activity. In contrast, enzymatic activity was retained after T560S exchange. Enzyme variants with strongly reduced activity were also obtained when we mutated Gln-294 (binding partner of Thr-560) and Asn-287 (binding partner of Gln-294 and Met-418) to Leu. Basic kinetic characterization of the T560M mutant indicated that the enzyme lacks a kinetic lag phase but is rapidly inactivated. These data suggest that the low catalytic efficiency of the naturally occurring T560M mutant is caused by alterations of a hydrogen bond network interconnecting this residue with active site constituents. Disturbance of this bonding network increases the susceptibility of the enzyme for suicidal inactivation.

scholarly journals A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase

JACS Au ◽  
2021 ◽  
Author(s):  
Mary Ortmayer ◽  
Florence J. Hardy ◽  
Matthew G. Quesne ◽  
Karl Fisher ◽  
Colin Levy ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Heme Peroxidase

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Kohei Sasamoto ◽  
Tomoki Himiyama ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Koichi Uegaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cellulose Acetate ◽  
Electron Density ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Packing ◽  
Signal Sequence ◽  
Industrial Applications ◽  
Side Chain ◽  
Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

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