Characterization of the general anion-binding site in glutamate dehydrogenase-NADPH complex

1991 ◽  
Vol 1073 (1) ◽  
pp. 18-22 ◽  
Author(s):  
R. Srinivasan
Keyword(s):  
Binding Site ◽  
Glutamate Dehydrogenase ◽  
Anion Binding

Characterization of the Allosteric Anion-Binding Site ofO-Acetylserine Sulfhydrylase†

Biochemistry ◽  
2001 ◽  
Vol 40 (25) ◽  
pp. 7446-7452 ◽  
Author(s):  
Chia-Hui Tai ◽  
Peter Burkhard ◽  
David Gani ◽  
Thierry Jenn ◽  
Corey Johnson ◽  
...  
Keyword(s):  
Binding Site ◽  
Anion Binding

Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations

2002 ◽  
Vol 363 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Jie FANG ◽  
Betty Y. L. HSU ◽  
Courtney M. MacMULLEN ◽  
Mortimer PONCZ ◽  
Thomas J. SMITH ◽  
...  
Keyword(s):  
Binding Site ◽  
Glutamate Dehydrogenase ◽  
Purification And Characterization ◽  
X Ray Crystallography ◽  
Gtp Binding ◽  
Regulatory Mutations ◽  
Heterozygous Patient ◽  
Structural Mechanisms ◽  
Atp Regulation

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210μM) and S448P (ED50, 3.1μM) human GDH mutants compared with the wild-type human GDH (ED50, 42nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.

Characterization of the Fe(III) Binding Site in Sepia Eumelanin

2004 ◽  
Author(s):  
Alexander Samokhvalov ◽  
Yan Liu ◽  
John Simon
Keyword(s):  
Binding Site

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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