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 multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. 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 D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.
Qin
Y M
YM
Biocenter Oulu and Department of Biochemistry, University of Oulu, Box 3000, FIN-90014 Oulun Yliopisto, Finland.
Haapalainen
A M
AM
Kilpeläinen
S H
SH
Marttila
M S
MS
Koski
M K
MK
Glumoff
T
T
Novikov
D K
DK
Hiltunen
J K
JK
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020675:Peroxisomes / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D004746:Enoyl-CoA Hydratase; C083735:long-chain-enoyl-CoA hydratase