JBC, Vol. 251, Issue 17, 5352-5360, Sep, 1976
Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase
K. R. Srinivasan and S. Kumar
Structural and conformational organization of chicken liver fatty acid
synthetase has been probed using its fluorescent coenzyme, NADPH. Three
NADPH binding sites per mole of the enzyme complex, of apparently identical
dissociation constant (KD = 0.6 muM) can be titrated at temperatures above
12 degrees. These results are in disagreement with the earlier studies of
Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9,
245-251) in which four such sites could be titrated. At 12 degrees, the
composite sites split into two subsets: a pair of sites with a KD of 0.3
muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees
or 2 degrees), the site with weak affinity disappears, leaving a pair of
sites with a Kd of 0.5 muM. Similar observations were made when the enzyme
was modified with phenylmethylsulfonyl fluoride, a specific and selective
inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme
complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial
modification with phenylmethylsulfonyl fluoride elicits a NADPH binding
response similar to the binding observed at 12 degrees, i.e. two sets of
binding sites with nonidentical dissociation constants. Further
modification corresponding to the complete loss of deacylase function
results in a set of two apparently identical binding sites, and the third
site is not available for titration. The modified enzyme retains the two
reductase functions as measured by the model substrates,
acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition
of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the
NADPH binding affinity by a factor of 3. Other observations show that the
quantum yield, as measured by the ratio of fluorescence intensity of bound
and free NADPH, changes with temperature and ionic strength. Lowering the
temperature from 30 degrees to 2 degrees increases the enhancement ratio by
50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium
phosphate lowers it to 50% of the original level. Measurement of NADPH
binding in the presence of NADP+, NADH, NAD+ and
adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows
competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+
show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated
interactions implicating nonspecific conformational alteration of the
enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose
is intermediate between NADP+ and NADH. These data are discussed in terms
of substrate-mediated conformational changes and the moles of each of the
reductase enzymes per mole of the enzyme complex, the polarity of the NADPH
binding region, and the probable structure of the nicotinamide moiety when
bound to the enzyme.
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