Effects of substrate binding and pH on the secondary structure of carnitine acetyltransferase.
Carnitine acetyltransferase (CAT) exists as a monomer in solution as demonstrated by dynamic light scattering measurements. Under these conditions, interactions between CAT and its substrates, L-carnitine and acetyl-CoA, were studied by circular dichroism (CD) and fluorescence spectroscopy over a wide range of substrate concentrations. CD data indicated that the binding of L-carnitine and acetyl-CoA caused changes in the secondary structure of the protein. Quenching of the intrinsic protein fluorescence upon binding of either substrate corroborated these findings. Analysis of the binding data suggests that binding of both substrates to CAT is specific and saturable, and that there is a single binding site (or multiple identical and independent binding sites) on CAT for each substrate. Estimated L-carnitine/CAT dissociation constants were 506 +/- 58 microM and 236 +/- 27 microM in the absence or presence of acetyl-CoA, respectively. The dissociation constant for acetyl-CoA/CAT was estimated at 19 +/- 7 microM. The effect of pH on the secondary structure of the protein was determined in order to investigate the structural cause for the pH-dependent enzymatic activity of CAT. Loss of alpha-helices and a reduction of thermal stability in CAT was detected at both acidic and basic pH. Thus, the reduced catalytic activity of CAT at acidic or basic pH may be due to pH-induced protein unfolding.
Yan
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B
Department of Central Technologies Preclinical Research, Sandoz Research Institute, Sandoz Pharmaceuticals Corporation, East Hanover, NJ 07936, USA.
Mann
W R
WR
Bell
P A
PA
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002331:Carnitine / Q000378:metabolism; D002332:Carnitine O-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D010856:Columbidae; D006863:Hydrogen-Ion Concentration; D008027:Light; D009132:Muscles / Q000201:enzymology; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence
D012996:Solutions; D000105:Acetyl Coenzyme A; D002332:Carnitine O-Acetyltransferase; D002331:Carnitine