Role of active-site residues in the catalytic mechanism of ribonuclease A

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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The mechanistic role of active site residues in non-stereo haloacid dehalogenase E (DehE)

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2019.05.003 ◽

2019 ◽

Vol 90 ◽

pp. 219-225 ◽

Cited By ~ 3

Author(s):

Muhammad Hasanuddin Zainal Abidin ◽

Khairul Bariyyah Abd Halim ◽

Fahrul Huyop ◽

Tengku Haziyamin Tengku Abdul Hamid ◽

Roswanira Abdul Wahab ◽

...

Keyword(s):

Active Site ◽

Active Site Residues ◽

Haloacid Dehalogenase

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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Mutagenesis of Two Acidic Active Site Residues in Human Muscle Creatine Kinase: Implications for the Catalytic Mechanism†

Biochemistry ◽

10.1021/bi0020980 ◽

2001 ◽

Vol 40 (10) ◽

pp. 3056-3061 ◽

Cited By ~ 19

Author(s):

John S. Cantwell ◽

Walter R. Novak ◽

Pan-Fen Wang ◽

Michael J. McLeish ◽

George L. Kenyon ◽

...

Keyword(s):

Creatine Kinase ◽

Active Site ◽

Catalytic Mechanism ◽

Human Muscle ◽

Active Site Residues ◽

Muscle Creatine Kinase ◽

Muscle Creatine

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GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

Biochemical Journal ◽

10.1042/bj20090110 ◽

2009 ◽

Vol 420 (2) ◽

pp. 221-227 ◽

Cited By ~ 56

Author(s):

Helge C. Dorfmueller ◽

Vladimir S. Borodkin ◽

Marianne Schimpl ◽

Daan M. F. van Aalten

Keyword(s):

Active Site ◽

Cell Biology ◽

Rational Design ◽

Catalytic Mechanism ◽

Conserved Residues ◽

Cellular Processes ◽

Acetyl Group ◽

Nanomolar Range ◽

Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

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