Role of Glu51 for Cofactor Binding and Catalytic Activity in Pyruvate Decarboxylase from Yeast Studied by Site-Directed Mutagenesis†

Biochemistry ◽  
1997 ◽  
Vol 36 (7) ◽  
pp. 1900-1905 ◽  
Author(s):  
Margrit Killenberg-Jabs ◽  
Stephan König ◽  
Ines Eberhardt ◽  
Stefan Hohmann ◽  
Gerhard Hübner
Keyword(s):  
Catalytic Activity ◽  
Pyruvate Decarboxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cofactor Binding

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

Exploring the role of histidines in the catalytic activity of duck δ-Crystallins using site-directed mutagenesis

1995 ◽  
Vol 61 (2) ◽  
pp. 151-154 ◽  
Author(s):  
Gerald Patejunas ◽  
Peter Barbosa ◽  
Michelle Lacombe ◽  
William E. o'Brien
Keyword(s):  
Catalytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

The role of conserved residues in the catalytic activity of NDM-1: an approach involving site directed mutagenesis and molecular dynamics

2019 ◽  
Vol 21 (32) ◽  
pp. 17821-17835 ◽  
Author(s):  
Abid Ali ◽  
Rakesh Kumar ◽  
Mir Asif Iquebal ◽  
Sarika Jaiswal ◽  
Dinesh Kumar ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Catalytic Activity ◽  
Cell Survival ◽  
Conformational Changes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Conserved Residues

Drug degraded by enzyme and hence not targeted on to the cell leading to cell survival. After mutation leading to conformational changes and loss of function hence drug was not degraded and remained available for the target to lyse the cell.

scholarly journals Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

1992 ◽  
Vol 267 (36) ◽  
pp. 25754-25758
Author(s):  
J Wu ◽  
D Filer ◽  
A.J. Friedhoff ◽  
M Goldstein
Keyword(s):  
Tyrosine Hydroxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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