Role of glutamate 144 and glutamate 164 in the catalytic mechanism of enoyl-CoA hydratase.
The role of two glutamate residues (E164 and E144) in the active site of enoyl-CoA hydratase has been probed by site-directed mutagenesis. The catalytic activity of the E164Q and E144Q mutants has been determined using 3'-dephosphocrotonyl-CoA. Removal of the 3'-phosphate group reduces the affinity of the substrate for the enzyme, thereby facilitating the determination of K(m) and simplifying the analysis of the enzymes' pH dependence. k(cat) for the hydration of 3'-dephosphocrotonyl-CoA is reduced 7700-fold for the E144Q mutant and 630000-fold for the E164Q mutant, while K(m) is unaffected. These results indicate that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme. Previously, we reported that, in contrast to the wild-type enzyme, the E164Q mutant was unable to exchange the alpha-proton of butyryl-CoA with D(2)O [D'Ordine, R. L., Bahnson, B. J., Tonge, P. J. , and Anderson, V. E. (1994) Biochemistry 33, 14733-14742]. Here we demonstrate that E144Q is also unable to catalyze alpha-proton exchange even though E164, the glutamate that is positioned to abstract the alpha-proton, is intact in the active site. The catalytic function of each residue has been further investigated by exploring the ability of the wild-type and mutant enzymes to eliminate 2-mercaptobenzothiazole from 4-(2-benzothiazole)-4-thiabutanoyl-CoA (BTTB-CoA). As expected, reactivity toward BTTB-CoA is substantially reduced (690-fold) for the E164Q enzyme compared to wild-type. However, E144Q is also less active than wild-type (180-fold) even though elimination of 2-mercaptobenzothiazole (pK(a) 6.8) should require no assistance from an acid catalyst. Clearly, the ability of E164 to function as an acid-base in the active site is affected by mutation of E144 and it is concluded that the two glutamates act in concert to effect catalysis.
Department of Chemistry, SUNY at Stony Brook 11794-3400, USA.
H A
HA
Hofstein
Y
Y
Feng
V E
VE
Anderson
P J
PJ
Tonge
D000818:Animals; D052160:Benzothiazoles; D044926:Butyryl-CoA Dehydrogenase; D002384:Catalysis; D002934:Cinnamates / Q000737:chemistry; D003437:Crotonates / Q000737:chemistry; D018360:Crystallography, X-Ray; D004746:Enoyl-CoA Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044943:Fatty Acid Desaturases / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013844:Thiazoles / Q000737:chemistry
D052160:Benzothiazoles; D002934:Cinnamates; D003437:Crotonates; D007093:Imidazoles; D011994:Recombinant Proteins; D013844:Thiazoles; D018698:Glutamic Acid; C049259:(4-(dimethylamino)cinnamoyl)imidazole; C005563:captax; D044943:Fatty Acid Desaturases; D044926:Butyryl-CoA Dehydrogenase; D004746:Enoyl-CoA Hydratase