TY - JOUR
T1 - Glutamate 170 of Human L-3-Hydroxyacyl-CoA Dehydrogenase is Required for Proper Orientation of the Catalytic Histidine and Structural Integrity of the Enzyme
AU - Barycki, Joseph J.
AU - O'Brien, Laurie K.
AU - Strauss, Arnold W.
AU - Banaszak, Leonard J.
PY - 2001/9/28
Y1 - 2001/9/28
N2 - L-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the β-oxidation spiral, reversibly catalyzes the conversion of L-3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. Similar to other dehydrogenases, HAD contains a general acid/base, His158, which is within hydrogen bond distance of a carboxylate, Glu170. To investigate its function in this catalytic dyad, Glu170 was replaced with glutamine (E170Q), and the mutant enzyme was characterized. Whereas substrate and cofactor binding were unaffected by the mutation, E170Q exhibited diminished catalytic activity. Protonation of the catalytic histidine did not restore wild-type activity, indicating that modulation of the pKa of His158 is not the sole function of Glu170. The pH profile of charge transfer complex formation, an independent indicator of active site integrity, was unaltered by the amino acid substitution, but the intensity of the charge transfer band was diminished. This observation, coupled with significantly reduced enzymatic stability of the E170Q mutant, implicates Glu170 in maintenance of active site architecture. Examination of the crystal structure of E170Q in complex with NAD+ and acetoacetyl-CoA (R = 21.9%, Rfree = 27.6%, 2.2 A) reveals that Gln170 no longer hydrogen bonds to the side chain of His 158. Instead, the imidazole ring is nearly perpendicular to its placement in the comparable native complex and no longer positioned for efficient catalysis.
AB - L-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the β-oxidation spiral, reversibly catalyzes the conversion of L-3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. Similar to other dehydrogenases, HAD contains a general acid/base, His158, which is within hydrogen bond distance of a carboxylate, Glu170. To investigate its function in this catalytic dyad, Glu170 was replaced with glutamine (E170Q), and the mutant enzyme was characterized. Whereas substrate and cofactor binding were unaffected by the mutation, E170Q exhibited diminished catalytic activity. Protonation of the catalytic histidine did not restore wild-type activity, indicating that modulation of the pKa of His158 is not the sole function of Glu170. The pH profile of charge transfer complex formation, an independent indicator of active site integrity, was unaltered by the amino acid substitution, but the intensity of the charge transfer band was diminished. This observation, coupled with significantly reduced enzymatic stability of the E170Q mutant, implicates Glu170 in maintenance of active site architecture. Examination of the crystal structure of E170Q in complex with NAD+ and acetoacetyl-CoA (R = 21.9%, Rfree = 27.6%, 2.2 A) reveals that Gln170 no longer hydrogen bonds to the side chain of His 158. Instead, the imidazole ring is nearly perpendicular to its placement in the comparable native complex and no longer positioned for efficient catalysis.
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U2 - 10.1074/jbc.M104839200
DO - 10.1074/jbc.M104839200
M3 - Article
C2 - 11451959
AN - SCOPUS:0035965209
SN - 0021-9258
VL - 276
SP - 36718
EP - 36726
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 39
ER -