Glutamate 170 of human L-3-hydroxyacyl-CoA dehydrogenase is required for proper orientation of the catalytic histidine and structural integrity of the enzyme

L-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the beta -oxidation spiral, reversibly catalyzes the conversion Of L-3-hydroxyacyl- CoA to the corresponding 3-ketoacyl-CoA. Similar to other dehy-drogenases, HAD contains a general acid/base, His(158), which is within hydrogen bond d istance of a carboxylate, Glu(170). To investigate its function in this cat alytic dyad, Glu(170) was replaced with glutamine (E170Q), and the mutant e nzyme was characterized. Whereas substrate and cofactor binding were unaffe cted by the mutation, E170Q exhibited diminished catalytic activity. Proton ation of the catalytic histidine did not restore wild-type activity, indica ting that modulation of the pK(a) of His(158) is not the sole function of G lu(170). The pH profile of charge transfer complex formation, an independen t indicator of active site integrity, was unaltered by the amino acid subst itution, but the intensity of the charge transfer band was diminished. This observation, coupled with significantly reduced enzymatic stability of the E170Q mutant, implicates Glu(170) in maintenance of active site architectu re. Examination of the crystal structure of E170Q in complex with NAD(+) an d aceto-acetyl-CoA (R = 21.9%, R-free, = 27.6%, 2.2 Angstrom) reveals that Gln(170) no longer hydrogen bonds to the side chain of His(158). Instead, t he imidazole ring is nearly perpendicular to its placement in the comparabl e native complex and no longer positioned for efficient catalysis.