Multiple Active Site Conformations Revealed by Distant Site Mutation in Ornithine Decarboxylase†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (41) ◽  
pp. 12990-12999 ◽  
Author(s):  
Laurie K. Jackson ◽  
Jeffrey Baldwin ◽  
Radha Akella ◽  
Elizabeth J. Goldsmith ◽  
Margaret A. Phillips
Keyword(s):  
Ornithine Decarboxylase ◽  
Active Site ◽  
Site Mutation ◽  
Distant Site

scholarly journals Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 985-994 ◽  
Author(s):  
Jin Kyun Kim ◽  
Cheol Lee ◽  
Seon Woo Lim ◽  
Jacob T. Andring ◽  
Aniruddha Adhikari ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Kinetic Parameters ◽  
Active Site ◽  
Systematic Approach ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Site Mutation ◽  
Functional Changes ◽  
Reaction Rate Constants

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 −) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.

scholarly journals Active Site Mutation A126G Abrogates PI(4,5)P2-Mediated Allosteric Activation of the Tumor Suppressor PTEN

2019 ◽  
Vol 116 (3) ◽  
pp. 368a
Author(s):  
Caroline Zedler ◽  
Sven-Andreas Freibert ◽  
Christian R. Halaszovich ◽  
Dominik Oliver ◽  
Kirstin Hobiger
Keyword(s):  
Tumor Suppressor ◽  
Active Site ◽  
Site Mutation ◽  
Allosteric Activation

scholarly journals Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation

1999 ◽  
Vol 274 (44) ◽  
pp. 31203-31208 ◽  
Author(s):  
Rachel Graber ◽  
Patrik Kasper ◽  
Vladimir N. Malashkevich ◽  
Pavel Strop ◽  
Heinz Gehring ◽  
...  
Keyword(s):  
Active Site ◽  
Aspartate Aminotransferase ◽  
Site Mutation

Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

FEBS Journal ◽  
2012 ◽  
Vol 279 (10) ◽  
pp. 1777-1787 ◽  
Author(s):  
Tine Skovgaard ◽  
Ulla Uhlin ◽  
Birgitte Munch-Petersen
Keyword(s):  
Caenorhabditis Elegans ◽  
Thymidine Kinase ◽  
Active Site ◽  
Thymidine Kinase 1 ◽  
Site Mutation

Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine

2001 ◽  
Vol 360 (3) ◽  
pp. 657-665 ◽  
Author(s):  
Yong-Sun LEE ◽  
Young-Dong CHO
Keyword(s):  
Ornithine Decarboxylase ◽  
Active Site ◽  
Competitive Inhibition ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Three Dimensional Model ◽  
Nicotiana Glutinosa ◽  
Polyamine Biosynthesis ◽  
Conserved Sequence

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank® AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native Mr of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with Km values of 562μM and 1592μM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (Ki 1.15μM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys95) and cysteine (Cys96, Cys338 and Cys377) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys96, each mutation caused a substantial loss in enzyme activity. Most notably, Lys95 increased the Km for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the kcat for ODC and lysine decarboxylase (LDC) activity respectively. The Cys377 → Ala mutant possessed a kcat that was lowered by 23-fold, and the Km value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease 1 1Edited by P. E. Wright

1999 ◽  
Vol 287 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Tadahide Izumi ◽  
Jedrzej Malecki ◽  
M.Ahmad Chaudhry ◽  
Michael Weinfeld ◽  
Jeff H Hill ◽  
...  
Keyword(s):  
Active Site ◽  
Site Mutation
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