Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase.
Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.
Haas
Jeffrey A
JA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Fox
Brian G
BG
D000213:Acyl Carrier Protein / Q000737:chemistry; D000215:Acylation; D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D003619:Dansyl Compounds / Q000737:chemistry; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D005288:Ferredoxins / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D055672:Static Electricity; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D001059:Apoproteins; D003619:Dansyl Compounds; D029968:Escherichia coli Proteins; D005288:Ferredoxins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C040157:acpP protein, E coli; D010204:Pantetheine; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase; D054889:Fatty Acid Synthase, Type II; D016650:Fluorescein-5-isothiocyanate; C003129:4'-phosphopantetheine