Analysis of active site loop amino acids of lactate dehydrogenase fromPlasmodium vivaxby site-directed mutagenesis studies

2006 ◽  
Vol 67 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Dilek Turgut-Balik ◽  
Dilek Sadak ◽  
Venhar Celik
Keyword(s):  
Amino Acids ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

Site-Directed Mutagenesis of Firefly Luciferase Active Site Amino Acids:  A Proposed Model for Bioluminescence Color†

Biochemistry ◽  
1999 ◽  
Vol 38 (40) ◽  
pp. 13223-13230 ◽  
Author(s):  
Bruce R. Branchini ◽  
Rachelle A. Magyar ◽  
Martha H. Murtiashaw ◽  
Shannon M. Anderson ◽  
Lisa C. Helgerson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Proposed Model

Roles of Amino Acids in theEscherichia coliOctaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis

Biochemistry ◽  
2012 ◽  
Vol 51 (16) ◽  
pp. 3412-3419 ◽  
Author(s):  
Keng-Ming Chang ◽  
Shih-Hsun Chen ◽  
Chih-Jung Kuo ◽  
Chi-Kang Chang ◽  
Rey-Ting Guo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-Directed Mutagenesis of Amino Acids Associated with the Active Site of Rubisco from Anacystis Nidulans

1990 ◽  
pp. 2257-2260
Author(s):  
M. A. Parry ◽  
C. A. Kettleborough ◽  
N. Halford ◽  
A. L. Phillips ◽  
R. Branden ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Anacystis Nidulans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Asymmetry in catalysis: ‘unidirectional’ amino acid racemases

2021 ◽  
Vol 43 (1) ◽  
pp. 28-34
Author(s):  
Stephen L. Bearne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Hydroxyl Group ◽  
Directed Mutagenesis ◽  
Biotechnological Applications ◽  
Enzyme Substrate ◽  
Brønsted Base ◽  
Catalytic Dyad

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed industrially in biotransformation reactions. Despite their enzyme–substrate complexes being diastereomers, some racemases and epimerases exhibit a kinetic pseudo-symmetry, binding their enantiomeric or epimeric substrate pairs with roughly equal affinities and catalyzing their stereoinversion with similar turnover numbers. In other cases, this kinetic pseudo-symmetry is absent or may be ‘broken’ by substitution of a catalytic Cys by Ser at the active site of cofactor-independent racemases and epimerases, or by altering the Brønsted base of the catalytic dyad that facilitates deprotonation of the Cys residue. Moreover, a natural Thr-containing l-Asp/Glu racemase was discovered that catalyses ‘unidirectional’ substrate turnover, unlike the typical bidirectional racemases and epimerases. These observations suggest that bidirectional Cys–Cys racemases may be re-engineered into ‘unidirectional’ racemases through substitution of the thiol by a hydroxyl group. Catalysis by such ‘unidirectional’ racemase precursors could then be optimized further by site-directed mutagenesis and directed evolution to furnish useful enzymes for biotechnological applications.

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