Static curvature and flexibility measurements of DNA with microscopy. A simple renormalization method, its assessment by experiment and simulation.

We present the derivation of equations based on statistical polymer chain analysis and a method to quantify the average angle value of intrinsic bends and the local flexibility at a given locus on DNA fragments imaged by electron microscopy. DNA fragments of n base-pairs are considered as stiff chains of n jointed unit rigid rods. If the DNA fragments are composed of two branches A0Am and A0Bn, with, respectively, m and n base-pairs, where the standard deviations of the angle formed by two consecutive base-pairs are uniform over each branch, respectively, sigmathetaA and sigmathetaB, we show that the standard deviation of the angle AmA0Bn is: [formula: see text] where sigmatheta0 is the standard deviation of the angle at locus A0. This equation is established for small angular deviations by analysis of DNA at different scales and the validity of the methodology is controlled with the computation of the reduced chi2 statistical test. The length of the DNA fragments must be of the order of, or below, the persistence length, as determined by sets of statistics from computer simulations of DNA fragments. This is verified experimentally by a detailed analysis of the digitized contours of homogeneous linear 139 base-pair DNA fragments observed by electron microscopy. The images are compared to the reconstruction of DNA fragments from the measurements. The value found, sigma0=4.6 degrees/bp, is consistent with the well-accepted value for DNA in a plane. We discuss the relationship between the standard deviation of the measured angles and the flexibility at the base-pair level. This method is useful to quantify directly from microscopy techniques, such as electron or scanning force microscopy, the true bending angle, either intrinsic or induced by a ligand, and its associated flexibility at a given locus in any small DNA fragment.

DNA bending induced by the archaebacterial histone-like protein MC1.

The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1. This model amounts to double-folded Gaussian distributions and yields an equilibrium deflection angle of theta0=116 degrees for the DNA with bound MC1. It allowed measurements of the fraction of DNA with bound MC1 to be taken as a function of MC1 concentrations and yields an equilibrium dissociation constant of Kd=100 nM. It shows that the flexibility of DNA is reduced by the binding of MC1 and the formation of a kink. The equilibrium dissociation constant value was corroborated by gel electrophoresis. Control of the model by the computation of the reduced chi2 shows that the measurements are consistent and that electron microscopy can be used to quantify precisely the DNA deformations induced by the binding of a protein to a preferential site.

Solution structure of N-(2-deoxy-D-erythro-pentofuranosyl)urea frameshifts, one intrahelical and the other extrahelical, by nuclear magnetic resonance and molecular dynamics.

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.

Solution structure as a function of pH of two central mismatches, C . T and C . C, in the 29 to 39 K-ras gene sequence, by nuclear magnetic resonance and molecular dynamics.

The DNA duplexes 5' d(GCCACCAGCTC) x d(GAGCTXGTGGC), where the base X is either cytosine or thymine, have been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The sequence studied corresponds to the region 29 to 39 of the K-ras gene and is a hot spot for mutations. The results show that both duplexes adopt a globally B-DNA-type structure. For the C x C mismatch, we observe a structural change as a function of pH with an apparent pK of 6.95. The neutral species has only one hydrogen bond between the two bases but shows two families of wobble structures where one base or the other is displaced in the major groove. The protonated species has two hydrogen bonds and two structures but of unequal populations. In both systems, the sugar puckers remain predominantly C2'-endo and no significant changes in the backbone structure are observed. The neutral C . T mismatch is stabilized by two hydrogen bonds but, surprisingly, it can also be protonated, although the apparent pK is much lower, 5.65. In this case, protonation does not result in an additional hydrogen bond but must be due to better base-stacking interactions for C+ x T. The NMR data show that the environment of the T imino proton is very similar for C x T and C+ x T, although the hydrogen bond acceptor would be expected to be a nitrogen atom in the former case and an oxygen atom in the latter. We propose that for both structures there is an intervening water molecule which in addition reduces backbone strain. We have also measured the fluctuations during molecular dynamics runs in these mismatches. All are greater than for Watson-Crick base-pairs and the C x C mismatch shows very pronounced mobility.

Solution structure of two mismatches G.G and I.I in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

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.

Solution structure of two mismatches A.A and T.T in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

Two mismatches, one homopurine (A.A) and the other homopyrimidine (T.T), have been incorporated at the central position N of: 5'd(GCCACNAGCTC).d(GAGCTNGTGGC) in order to study nuclear magnetic resonance spectra and molecular dynamics. These duplexes constitute the sequence 29-39 of the K-ras gene coding for Gly12, 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 both systems, no structural change is observed in the pH range 4.7-9. For the duplex containing the homopurine A.A mismatch, we propose a type of pairing involving one hydrogen bond between the amino group of one central adenine and the nitrogen N1 of the opposite adenine. For the duplex containing the mispaired T.T bases, NMR spectra recorded in H2O at 282 K indicate that these central bases are engaged in wobble pairing, involving two imino-carbonyl hydrogen bonds. For both systems two conformations with the same donor and acceptor pattern can coexist, one being 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 for the T.T mispair and also inferred from NMR measurements on the A.A system. The presence of this exchange and its pathway has been investigated by molecular dynamics calculations on both systems. Distance restrained and unrestrained molecular dynamics are in very good agreement with the NMR data. The average structure for either mispair shows only small conformational change from normal B DNA. For each, a systematic pathway is observed for exchange between the two conformations.

Solution structure of an oncogenic DNA duplex, the K-ras gene and the sequence containing a central C.A or A.G mismatch as a function of pH: nuclear magnetic resonance and molecular dynamics studies.

The DNA duplex 5' d(GCCACCAGCTC)-d(GAGCTGGTGGC) corresponds to the sequence 29 to 39 of the K-ras gene, which contains a hot spot for mutations. This has been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The results show that it adopts a globally B-DNA type structure. We have introduced, at the central base-pair, the mismatches C.A and A.G. The mismatch position is that of the first base of the Gly12 codon, the hot spot. For the C.A mismatch we observe a structural change as a function of pH with an apparent pKa of 7.2. At low pH, the mismatch pair adopts a structure close to a classic wobble conformation with the cytidine residue displaced into the major groove. It is stabilised by two hydrogen bonds in which the adenosine residue is protonated and the cytidine residue has a significant C3'-endo population. At high pH, the mispair structure is in equilibrium between wobble and reverse wobble conformations. Similar studies are reported on the A.G mismatch, which also undergoes a transition as a function of pH. 31P spectra have been recorded on all systems and as a function of pH. No evidence for BII phosphodiester backbone conformations was found. The NMR results are well corroborated by molecular dynamics calculations performed with or without distance constraints. The dynamics at the mismatch sites have been examined. Although the overall structures are close to B-DNA, helical parameters fluctuate differently at these sites. Different hydrogen bonding alternatives in dynamic equilibrium that can involve three-centred hydrogen bonds are observed.

Helical parameters, fluctuations, alternative hydrogen bonding, and bending in oligonucleotides containing a mistmatched base-pair by NOESY distance restrained and distance free molecular dynamics.

We have analysed and compared the molecular structures and dynamics of the DNA duplex, that corresponds to the sequence 29 to 39 of the K-ras gene, where the central base-pair is the normal C.G base or a mismatch base-pair C.A, C.A+, A.G and A+.G. Molecular dynamics runs of 100 picoseconds without or with distance restraints derived from NOE measurements are analysed and compared in all cases. (1) The EMBO convention of helical parameters for nucleic acids is extended to account for the construction and the description of any DNA mismatched base-pair. (2) Both types of MD runs reproduce very well all NMR data, except the H8 H2 inter-residue distances where agreement is not as good. (3) Average parameter values and fluctuations are in good agreement with results derived from persistence length and torsion modulus measurements. (4) Our molecular dynamics suggest the presence, in certain cases, of three-centred hydrogen bonds, which should be viewed as an equilibrium between hydrogen-bonding alternatives. In the case of the C.A mismatch, we observe the correlation between transient DNA bending and possible hydrogen bonding between a base and its 5' neighbour on the opposite strand in the sequence CCA. (5) These molecular dynamics analyses and observations provide a coherent view for the results obtained from recent DNA crystal structures and for results derived from other techniques such as gel electrophoresis at C.A steps.

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics.

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC+, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1-4) have been extended to triple helical polynucleotides.

Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase.

The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees).

Object Command Language: a formalism to build molecular models and to analyze structural parameters in macromolecules, with applications to nucleic acids.

We have written a programming language OCL (Object Command Language) to solve, in a general way, two recurring problems that arise during the construction of molecular models and during the geometrical characterization of macromolecules: how to move precisely and reproducibly any part of a molecular model in any user-defined local reference axes; and how to calculate standard or user-defined structural parameters that characterize the complex geometries of any macromolecule. OCL endows the user with three main capabilities: the definition of subsets of the macromolecule, called objects in OCL, with a formalism from elementary set theory or lexical analysis; the definition of sequences of elementary geometrical operations, called procedures in OCL, enabling one to build arbitrary three-dimensional (3D) orthonormal reference frames, to be associated with previously defined objects; and the transmission of these definitions to programs that allow one to display, to modify and to analyze interactively the molecular structure, or to programs that perform energy minimizations or molecular dynamics. Several applications to nucleic acids are presented.

The pH dependent configurations of the C.A mispair in DNA.

The structure of the cytosine-adenine mispair in a 7 base pair duplex has been investigated by proton NMR spectroscopy. At low pH, the predominant structure is protonated on the A residue and assumes a wobble conformation consistent with previous findings. The C residue of the mispair is found in a C2'-C3' endo equilibrium. This is confirmed by molecular dynamics calculations which suggest that the conformation of the protonated wobble is flexible and not as rigid as a normal base pair. As the solution pH is raised, a structural transition is observed with an apparent pK of 7.54 at 23 degrees C. At higher pH the predominant structure is one in which both the C and A residues are intrahelical. Evidence is presented that this structure corresponds to a reverse wobble in which the two bases are held together by one hydrogen bond. This structure is much less stable than the protonated form and even at low temperature single strands are observed in slow exchange with the neutral duplex form.

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data.

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.

Abasic frameshift in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-self-complementary oligodeoxynucleotide duplex that contains a model abasic site. The duplex contains six GC base pairs plus the abasic site at the center of one strand and corresponds to an abasic frameshift. Two-dimensional NMR studies on the nonexchangeable protons show that the guanine bases on either side of the abasic site are stacked over each other and that the abasic site is rotated out of the helix. Close proton-proton interactions are observed between the H4' proton of the abasic site and sugar protons of the guanosine in the 5' direction, which allows the position of the free sugar to be well-defined. NOE buildup curves from NOESY spectra recorded at very short mixing times were used to calculate a set of interproton distances. This data set was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two conformations that differ in the sugar conformation of the guanosine next to the abasic site in the 3' direction were necessary to fit all the NMR data. One of these two conformations could only be stabilized by addition of counterions at specific sites.

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase.

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)

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme.

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)

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: acetylation-deacetylation.

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.