Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Biochemistry ◽  
1993 ◽  
Vol 32 (47) ◽  
pp. 12730-12735 ◽  
Author(s):  
Roman Sakowicz ◽  
Helmut K. W. Kallwass ◽  
Wendy Parris ◽  
Cyril M. Kay ◽  
J. Bryan Jones ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Substrate Binding

The importance of arginine 171 in substrate binding by Bacillus stearothermophilus lactate dehydrogenase

1987 ◽  
Vol 146 (1) ◽  
pp. 346-353 ◽  
Author(s):  
Keith W. Hart ◽  
Anthony R. Clarke ◽  
Dale B. Wigley ◽  
William N. Chia ◽  
David A. Barstow ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Substrate Binding

scholarly journals Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase

1994 ◽  
Vol 300 (2) ◽  
pp. 491-499 ◽  
Author(s):  
T J Nobbs ◽  
A Cortés ◽  
J L Gelpi ◽  
J J Holbrook ◽  
T Atkinson ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Mutant Enzyme ◽  
Hydroxyl Group ◽  
Side Chain ◽  
General Effect ◽  
Carboxylate Group ◽  
Wild Type

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Site-directed mutagenesis of Bacillus stearothermophilus lactate dehydrogenase

1987 ◽  
Vol 15 (1) ◽  
pp. 152-153 ◽  
Author(s):  
ANTHONY R. CLARKE ◽  
DALE B. WIGLEY ◽  
DAVID A. BARSTOW ◽  
WILLIAM N. CHIA ◽  
ADAM D. B. WALDMAN ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis

2015 ◽  
Vol 418 ◽  
pp. 50-56 ◽  
Author(s):  
Shijia Liu ◽  
Shangjin Shao ◽  
Linlin Li ◽  
Zhi Cheng ◽  
Li Tian ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Binding Specificity ◽  
Architecture Analysis

scholarly journals 1P-121 Roles of Asp297 in the vicinity of the active site of cytochrome P450cam in substrate binding(The 46th Annual Meeting of the Biophysical Society of Japan)

2008 ◽  
Vol 48 (supplement) ◽  
pp. S40
Author(s):  
Keisuke Sakurai ◽  
Katsuyoshi Harada ◽  
Kunitoshi Shimokata ◽  
Takashi Hayashi ◽  
Hideo Shimada
Keyword(s):  
Annual Meeting ◽  
Active Site ◽  
Substrate Binding ◽  
Cytochrome P450cam ◽  
Biophysical Society
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