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Publication Abstract from PubMed

A strictly conserved active site arginine residue (alphaR157) and two histidine residues (alphaH80 and alphaH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the alphaR157A mutant, the residual activity (k cat = 10 +/- 2 s(-1)) accounts for less than 1 % of the wild-type activity (k cat = 1100 +/- 30 s(-1)) while the K m value is nearly unchanged at 205 +/- 10 mM. On the other hand, mutation of the active site pocket alphaH80 and alphaH81 residues to alanine resulted in enzymes with k cat values of 220 +/- 40 and 77 +/- 13 s(-1), respectively, and K m values of 187 +/- 11 and 179 +/- 18 mM. The double mutant (alphaH80A/alphaH81A) was also prepared and provided an enzyme with a k cat value of 132 +/- 3 s(-1) and a K m value of 213 +/- 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the alphaH80A/alphaH81A, alphaH80W/alphaH81W, and alphaR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 A resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the alphaCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.,Martinez S, Wu R, Krzywda K, Opalka V, Chan H, Liu D, Holz RC J Biol Inorg Chem. 2015 Jul;20(5):885-94. doi: 10.1007/s00775-015-1273-3. Epub, 2015 Jun 16. PMID:26077812^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.