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The phosphatidylcholine (PC)-preferring phospholipase C (PLC) from Bacillus cereus (PLC(Bc)) hydrolyzes various 1,2-diacyl derivatives of PC at different rates. Substrates with side chains having eight or more carbons are present in micellular form in aqueous media and are processed most rapidly. The catalytic efficiency (k(cat)/K(m)) for the hydrolyses of short-chain PCs at concentrations below their respective critical micelle concentrations also decreases as the side chains become shorter, and this loss of efficiency owes its origin to increases in K(m). In order to ascertain whether the observed increases in K(m) might arise from conformational changes in the glycerol backbone, nuclear magnetic resonance (NMR) experiments were performed in D(2)O to determine the (3)J(HH) and (3)J(CH) coupling constants along the glycerol subunit of 1, 2-dipropanoyl-sn-glycero-3-phosphocholine (K(m)=61 mM), 1, 2-dibutanoyl-sn-glycero-3-phosphocholine (K(m)=21.2 mM) and 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (K(m)=2.4 mM). Using these coupling constants, the fractional populations for each rotamer about the backbone of each of substrate were calculated. Two rotamers, which were approximately equally populated, about the sn-1-sn-2 bond of each substrate were significantly preferred, and in these conformers, the oxygens on the sn-1 and sn-2 carbons of the backbone were synclinal to optimize intramolecular hydrophobic interactions between the acyl side chains. There was greater flexibility about the sn-2-sn-3 bond, and each of the three possible staggered conformations was significantly populated, although there was a slight preference for the rotamer in which the oxygen bearing the phosphate head group was synclinal to the oxygen at the sn-2 carbon and to the sn-1 carbon; in this orientation, the head group is folded back relative to the side chains. These studies demonstrate that there is no significant change in the conformation about the glycerol backbone as a function of side chain length in short-chain phospholipids. Thus, prior organization of the substrate seems an unlikely determinant of the catalytic efficiency of PLC(Bc), and other factors such as hydrophobic interactions or differential solvation/desolvation effects associated with the complexation of the substrate with PLC(Bc) may be involved.