The relationship between dental disease and cerebral vascular accident in elderly United States veterans

Enantiomers of N-methyl-N,alpha-methylbenzylbutyramide (1), 1-butyl-3-methyl-3'-alpha-methylbenzylurea (2), 1,2,3, 4-tetrahydro-1-naphthyl-N-butylcarbamate (3), 1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (4), 1, 1'-bi-2-naphthyl-2-ol-2'-N-butylcarbamate (5), and 1, 1'-bi-2-naphthyl-2-butyrate-2'-N-butylcarbamate (6) are inhibitors of porcine pancreatic cholesterol esterase-catalyzed hydrolysis of 4-nitrophenyl butyrate and of electric eel acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine in the presence of 5,5'-dithiobis-2-nitrobenzoate. For competitive inhibitors, values of the inhibition constant (Ki) and the enantiomeric ratio (Ecomp.) are investigated. For active site-directed irreversible inhibitors, values of the inhibition constant (Ki), the carbamylation constant (k2), the bimolecular rate constant (ki), and the enantiomeric ratio (E) are investigated. Toward both enzymes, compounds 1 are poor competitive inhibitors (Ki=102-104 microM) but have good enantioselectivities (Ecomp.=10-50, the preference for R). R-2 and S-2 are competitive inhibitors of acetylcholinesterase with Ki=26 and 80 microM, respectively (the preference for R) but are active site-directed irreversible inhibitors of cholesterol esterase with ki=4 and 16 M-1 sec-1, respectively (the preference for S). For those competitive inhibitions, both leaving group hydrophilic and hydrophobic binding sites of cholesterol esterase or both anionic substrate binding site and peripheral anionic binding site of acetylcholinesterase bind to N,N-methyl-alpha-methylbenzyl disubstituted amide parts of these inhibitors and the enzyme does not catalyze the hydrolysis of these inhibitors. The opposite stereopreference (S) for the inhibition of cholesterol esterase by compounds 2 may be due to the fact that N, N-methyl-alpha-methylbenzyl disubstituted amide parts of these inhibitors bind to the alkyl chain binding site of the enzyme. Compounds 3-6 are active site-directed irreversible inhibitors of cholesterol esterase (ki=1-13000 M-1 s-1) and peripheral anionic binding site-directed irreversible inhibitors of acetylcholinesterase (ki=1.7-1300 M-1 s-1). Compounds 3 have low enantioselectivities (E=1.3-1.4) for both enzymes. The stereopreference for atropisomers 4 and 6 is S-form toward both enzymes (E=2-30) and is identical to that of cholesterol esterase-catalyzed hydrolysis of 1,1'-bi-2-naphthyl-2,2'-diacylate. This stereopreference (S) may be due to the fact that the butyryl group or one of two butylcarbamate groups of S-atropisomers binds more effectively to the leaving group hydrophobic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase than that of R-atropisomers. The opposite stereopreference (R) for atropisomers 5 toward both enzymes may be due to a favorable interaction between the hydroxyl group of the inhibitors and the leaving group hydrophilic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase.     

Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect

Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions were simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed KS values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.     

Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.     

Monoclonal antibody AE-2 modulates carbamate and organophosphate inhibition of fetal bovine serum acetylcholinesterase

The monoclonal antibody AE-2, raised against the human erythrocyte acetylcholinesterase (AChE) dimer (acetylcholine acetylhydrolase, EC 3.1.1.7), binds to other mammalian AChEs, including the tetramer that occurs in fetal bovine serum (FBS). AE-2 partially inhibited the rate of hydrolysis of the charged substrate acetylthiocholine by FBS AChE, whereas it increased the rate of hydrolysis of the neutral substrate indophenyl acetate. Present results show that AE-2 decreases the rate of inhibition of FBS AChE by the positively charged organophosphate amiton-p-toluene sulfonate and the positively charged carbamates pyridostigmine and neostigmine but accelerates inhibition of FBS AChE by the neutral organophosphates paraoxon and diisopropylfluorophosphate. Results suggest that AE-2 may allosterically modulate an anionic site in the catalytic center of FBS AChE.     

Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.     

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.     

Catalytic significance of the specificity of divalent cations as KS* and kcat* cofactors for secreted phospholipase A2

Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant for the interfacial binary (E*C) and ternary (E*CL) complexes of PLA2 and substrate mimics (L). Of the cations(II) that promote the binding of a mimic to the enzyme at the interface (E*), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary E*CL complexes with kcat* of <1 s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second 18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of E*C and E*CL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn. These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that the higher ground state stability of the E*CS complex with the inhibitory cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen facilitates the near-attack geometry in the E*CaS, and the His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.     

Purification and kinetic characterisation of juvenile hormone esterase from Drosophila melanogaster

Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The KM and Vmax of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a KM of 120 micro M and a Vmax of at least 70 mumol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHP3), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB3 including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E, 3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Co-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH.     

Toxicological effects of beryllium on platelets and vascular endothelium

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.     

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.     

FSBA modifies both alpha- and beta-subunits of F1 specifically and can be bound together with AXP at the same alpha-subunit

Binding of 1 mole 5'-fluorosulfonylbenzoyladenosine (FSBA) per mol F1 induces about 50% inhibition of ATPase activity and 80% inhibition of ITPase activity. The binding of additional ligand results in a further inhibition of both activities. Maximally 5 mol/mol F1, causing complete inhibition of activity, can be bound. Using radioactive FSBA more label is found on alpha-subunits than on beta-subunits under the usual buffer conditions. The modified amino acids are alpha-Tyr300, alpha-Tyr244 and beta-Tyr368. Binding of FSBA, at least up to 3 mol/mol F1, does not result in loss of bound ADP, whether the starting enzyme contains 2, 3 or 4 bound nucleotides. Added adenine nucleotides compete with FSBA only for binding that results in modification of beta-subunits, shifting the alpha/beta ratio of bound label to higher values. It is concluded that the alpha-subunits contain two hydrophobic pockets for the binding of nucleoside moieties, with a different orientation relative to the P-loop. One pocket contains alpha-Tyr244 and alpha-Tyr300, the other beta-Tyr368. Since, however, in the binding of adenine nucleotide di- or triphosphates the P-loop is involved, only one of these ligands can bind per subunit. The previously not understood binding characteristics of several substrate analogues have now become interpretable on the assumption that also the structurally homologous beta-subunits contain 2 pockets where nucleoside moieties can bind. The kinetic effects of FSBA binding indicate that the first FSBA binds at the regulatory site that has a high affinity for ADP and pyrophosphate. Binding of pyrophosphate at this high-affinity regulatory site increases the Vmax of the enzyme, while binding at a second regulatory site, a low-affinity site, increases the rate of binding of FSBA with a factor of about 3. Binding of bicarbonate at this latter site is responsible for the disappearance of the apparent negative cooperativity of the ATPase activity.     

Proteinase K from the mold Tritirachium album Limber. Specificity and mode of action

1) The specificity of proteinase K towards amino acid and oligopeptide nitroanilide substrates is investigated. 2) The active center of the enzyme contains an extended binding region consisting of several subsites. An integral part of the S1-subsite are hydrophobic areas which were investigated by systematic elongation of the carbon skeleton in carboxylic acid 4-nitrophenyl esters. On the basis of these studies, a possible model of the S1-binding site is proposed. 3) Kinetic parameters for the hydrolysis of substituted phenyl acetates catalyzed by proteinase K have been measured at pH 7 and 25 degrees C. Deacylation of an acyl-enzyme intermediate is probably the rate-limiting step. Acylation (kcat/km used as a measure) is modestly sensitive to the sigma values of the substituents (p = 1.33, r = 0.9108), indicating electrophilic assistance by the enzyme in the catalytic mechanism. 4) Hydrophobic forces apparently are not involved in the binding of the leaving group.     

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.     

Endoscopic sclerotherapy is useful in Dieulafoy''s disease

1.5% Capsaicin (Cap) or Vehicle was respectively used to treat the right or left sciatic nerve in 20 Sprague-Dawley rats. On the seventh day, the 20 rats were at random divided into electroacupuncture (EA) group and non-EA group, the spinal cord corresponding to the afferent segments of sciatic nerve was taken out for observing the changes of acetylcholinesterase (AChE) activity and [3H]-quinuelidinylbenzylate (QNB) binding sites in the spinal dorsal horn (SDH). The results were as follows: (1) EA "Huantiao" could enhance AChE activity in the SDH and decrease [3H]-QNB binding sites; (2) Cap treating sciatice nerve could weaken AChE activity in the SDH and merease [3H]-QNB binding sites; (3) Cap treatment could inhibit or partially inhibit the actions of EA as above. The results indicated that ACh participated in the primary afferent of acupuncture information and might exist in Cap-sensitive neurons.     

Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Br?nsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Br?nsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.     

Differential effects of CD28 costimulation on HIV production by CD4+ cells

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.     

Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.     

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.     

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).     

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.