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The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

Two mismatches, G.G and I.I, have been incorporated at the central position of 5'd-(GCCACXAGCTC).d(GAGCTXGTGGC) in order to carry out NMR and molecular dynamics studies. These duplexes constitute the sequence 29-39 of the K-ras gene coding for the glycine 12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For the duplex containing the G.G mismatch, we propose for the major species, a type of pairing involving one hydrogen bond between the imino group of one central guanine and the carbonyl group of the opposite guanine. Both bases are in an anti conformation. Two conformations, with the same donor and acceptor pattern can coexist, one is obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature. A minor species involving hydrogen bonding between the guanine amino group and the carbonyl group of the guanine on the opposite strand may also exist as shown by the molecular dynamics calculations. For the I.I mismatch we observe the same major species, i.e., hydrogen bonding between an imino proton of one base and the carbonyl group of the base on the opposite strand with both bases in an anti conformation. Exchange between these two conformations is faster than for the G.G mismatch. Further, we observe that the I.I mismatch adopts a minor conformation, in which one or other of the bases is in the syn conformation.

The kinetics of the reaction of acetyl coenzyme A (AcCoA) with fatty acid synthase has been studied with a modified quenched-flow technique in 0.1 M potassium phosphate (pH 7.0), 0.5 mM ethylenediaminetetraacetic acid, and 10% glycerol (w/v) at 23 degrees C. The kinetics of the deacetylation of the isolated acetylated enzyme by CoA also was studied. An overall mechanism consistent with the data is (formula; see text) where E represents the enzyme. The equilibrium dissociation constants, K1 and K3, were estimated to be 85 and 70 microM, respectively, and the rate constants k2 and k-2 are 43 and 103 s-1, respectively. The maximum number of acetyl groups bound to the enzyme in terms of this mechanism is 3.8 (mol/mol). This mechanism also is consistent with the amount of acetylated enzyme formed during titrations of the enzyme and radioactive AcCoA with CoA. The spontaneous hydrolysis of the enzyme at 23 degrees C has a rate constant of 4.7 X 10(-4) s-1. The acetyl groups on the native enzyme are rapidly removed by hydroxylamine. However, 0.39 of the acetyl groups remains after treatment with hydroxylamine if the enzyme is first denatured in 4 M urea. This suggests that the acyl binding sites on the native enzyme are an unstable acetyl oxygen ester and an acetyl thio ester. Destruction of the thioesterase activity of the enzyme through chemical modification of the enzyme does not alter the rate of spontaneous hydrolysis of the acetyl-enzyme nor its reactivity toward hydroxylamine.

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding to fatty acid synthase from chicken liver and of the reduction of enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase) and the steps leading to formation of the acetoacetyl-enzyme have been studied in 0.1 M potassium phosphate-1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, at 25 degrees C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection has permitted the use of NADPH concentrations as low as 20 nM. The kinetics of the binding of NADPH to the enzyme is consistent with a simple bimolecular binding mechanism and four equivalent sites on the enzyme (presumably two beta-ketoacyl reductase sites and two enoyl reductase sites). The bimolecular rate constant is 12.7 X 10(6) M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium dissociation constant of 6.0 microM. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as two consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] much greater than [enzyme] much greater than [NADPH]. From the dependence of the rate of reduction of aceto-acetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium dissociation constant for NADPH binding to the enzyme of 5.9 microM is obtained, and the rate constant for the reduction is 17.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)