Human peroxisomal multifunctional enzyme type 2 - Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

beta-Oxidation of acyl-CoAs in mammalian peroxisomes can occur via either m ultifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catal yze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxy acyl-CoA, but with opposite chiral specificity. Amino acid sequence alignme nt of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s r eveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-4 08, Tyr-410, Asp-490, Tyr-505, Asp-810, His-515, Asp-517, and His-532, To i nvestigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were te sted for complementation in a yeast. After additional screening, the wild t ype and noncomplementing E366A and D510A variants were expressed and charac terized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K-m value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510 A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif f ound in the hydratase 2 proteins. The data show that the hydratase 2 reacti on catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, su ggesting that their catalytic role may be equivalent to that of the two cat alytic residues of hydratase 1.