GARDASIL: prophylactic human papillomavirus vaccine development--from bench top to bed-side.

GARDASIL (Merck, Whitehouse Station, NJ) is a non-infectious recombinant, quadrivalent vaccine prepared from the highly purified virus-like particles (VLPs) of the major capsid proteins of human papillomavirus (HPV) types 6, 11, 16, and 18. GARDASIL is the first vaccine approved for use in women aged 9-26 years for the prevention of cervical cancer and genital warts, as well as vulvar and vaginal precancerous lesions. This report describes some of the key preclinical efforts, achievements in pharmaceutical development, in vivo animal evaluation, and clinical trial data.

Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis.

Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.

Gene therapy and metabolic engineering.

Gene therapy involves the introduction of normal, healthy genes into cells to correct the underlying cause of a wide variety of inherited and acquired diseases. Future progress in developing effective clinical protocols involving gene therapy for the treatment of cellular dysfunction associated with disease may incorporate metabolic engineering. Metabolic engineering can be applied to gene therapy for the successful identification of disease genes; elucidation of disease pathways; development of safe and efficient gene-delivery systems; and regulation and control of gene expression. Cystic fibrosis, cancer, and diabetes are reviewed as examples of diseases where gene therapy approaches are being studied.

Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.

The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase.

Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (phi C(2) = 0.98 +/- 0.06) and HETDP C(alpha)-L (phi C(alpha) = 1.01 +/- 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (phi = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] < [S]) conditions shows solvent discrimination tritium isotope effects that increase over the range (kH/kT)disc = 0.39 (single turnover) to 0.86 (ten turnovers). The 2-fold increase in the value of (kH/kT)disc for the [1-L]acetaldehyde product under steady-state compared to single-turnover conditions is attributed to a fractionation factor of phi 1 = 0.88 +/- 0.06 for the residue(s) involved in C(alpha)-hydron transfer to form HETDP. This provides evidence that catalysis of acetaldehyde formation by PDC involves specific protonation of both HETDP C(alpha)-L and TDP C(2)-L (phi 2 = 1.0 +/- 0.1) and requires at least two catalytic groups. Values of phi < or = 1 for protonation of TDP C(2)-L and HETDP C(alpha)-L provide no evidence that the exocyclic 4'-amino or -imino group (phi > or = 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.

Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes.

Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide: pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha 4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C isotope discrimination against [1,2-14C]acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.

Thiazolium C(2)-proton exchange: structure-reactivity correlations and the pKa of thiamin C(2)-H revisited.

Rate constants for C(2)-proton exchange from thiamin, N(1')-methylthiamin, and several 3-substituted-4-methylthiazolium ions catalyzed by D2O and deuterioxide ion were determined by 1H NMR at 30 degrees C and ionic strength 2.0 M. Values of pKa for the thiazolium ions, including thiamin itself, were found to be in the range pKa = 17-19; the pKa values for N(1')-protonated thiamin and free thiamin C(2)-H in H2O are 17.7 and 18.0, respectively. The pKa value for N(1')-protonated thiamin was calculated from the observed rate constant for the pD-independent reaction with D2O after correction for a secondary solvent deuterium isotope effect of kH2O/kD2O = 2.6. The pKa value for free thiamin was calculated from the rate constant for catalysis by OD- after correction by a factor of 3.3 = 8/2.4 for an 8-fold negative deviation of kOD from the Brønsted plot of slope 1.0 for general base catalysis and a secondary solvent isotope effect of kOD/kOH = 2.4. Values of k-a = 2 X 10(10) and 3 X 10(9) M-1 s-1 were assumed for diffusion-controlled protonation of the C(2) ylide in the reverse direction by H3O+ and H2O, respectively. The Hammett rho I value for the exchange reaction catalyzed by deuterioxide ion or D2O is 8.4 +/- 0.2. There is no positive deviation of the rate constants for free or N(1')-substituted thiamin analogues in either Hammett correlation. This shows that the aminopyrimidinyl group does not provide significant intramolecular catalysis of nonenzymic C(2)-proton removal in the coenzyme.

The systematic characterization by aqueous column chromatography of solutes which affect protein stability.

We have systematically characterized, by aqueous column chromatography on a size exclusion cross-linked dextran gel (Sephadex G-10), 12 solutes, 11 of which are known to affect protein stability. Six are chaotropes (water structure breakers) and destabilize proteins, while five are polar kosmotropes (polar water structure makers) and stabilize proteins. Analysis of the chromatographic behavior of these neutral (ethylene glycol, urea), positively charged (Tris, guanidine, as the hydrochloride salts) and negatively charged (SO2-4, HPO2-4, F-, Cl-, Br-, Cl3CCO-2, I-, SCN-, as the sodium salts, in order of elution) solutes at pH 7 as a function of sample concentration (up to 0.6 M), supporting electrolyte, and temperature yields four conclusions, based largely on the behavior of the anions. Chaotropes adsorb to the gel according to their position in the Hofmeister series, with the most chaotropic species adsorbing most strongly. ++Chaotropes adsorb to the gel less strongly in the presence of chaotropes (a salting in effect) and more strongly in the presence of polar kosmotropes (a salting out effect). Polar kosmotropes do not adsorb to the gel, and are sieved through the gel according to their position in the Hofmeister series, with the most kosmotropic species having the largest relative hydrodynamic radii. The hydrodynamic radii of polar kosmotropes is increased by chaotropes and decreased by polar kosmotropes. These results suggest that a chaotrope interacts with the first layer of immediately adjacent water molecules somewhat less strongly than would bulk water in its place; a polar kosmotrope, more strongly.

Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions.

We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E. coli cells using a 4-step 16-fold purification procedure, a yield of 60%. We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity. The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo. Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used. The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate). The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.

The Hofmeister effect and the behaviour of water at interfaces.

Starting from known properties of non-specific salt effects on the surface tension at an air-water interface, we propose the first general, detailed qualitative molecular mechanism for the origins of ion-specific (Hofmeister) effects on the surface potential difference at an air-water interface; this mechanism suggests a simple model for the behaviour of water at all interfaces (including water-solute interfaces), regardless of whether the non-aqueous component is neutral or charged, polar or non-polar. Specifically, water near an isolated interface is conceptually divided into three layers, each layer being I water-molecule thick. We propose that the solute determines the behaviour of the adjacent first interfacial water layer (I1); that the bulk solution determines the behaviour of the third interfacial water layer (I3), and that both I1 and I3 compete for hydrogen-bonding interactions with the intervening water layer (I2), which can be thought of as a transition layer. The model requires that a polar kosmotrope (polar water-structure maker) interact with I1 more strongly than would bulk water in its place; that a chaotrope (water-structure breaker) interact with I1 somewhat less strongly than would bulk water in its place; and that a non-polar kosmotrope (non-polar water-structure maker) interact with I1 much less strongly than would bulk water in its place. We introduce two simple new postulates to describe the behaviour of I1 water molecules in aqueous solution. The first, the 'relative competition' postulate, states that an I1 water molecule, in maximizing its free energy (--delta G), will favour those of its highly directional polar (hydrogen-bonding) interactions with its immediate neighbours for which the maximum pairwise enthalpy of interaction (--delta H) is greatest; that is, it will favour the strongest interactions. We describe such behaviour as 'compliant', since an I1 water molecule will continually adjust its position to maximize these strong interactions. Its behaviour towards its remaining immediate neighbours, with whom it interacts relatively weakly (but still favourably), we describe as 'recalcitrant', since it will be unable to adjust its position to maximize simultaneously these interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Dihydroorotase from Escherichia coli. Purification and characterization.

Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3], which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli. Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35%. The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000. The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions. Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands. The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution. At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.

Purification of aqueous ethylene glycol.

Reagent-grade ethylene glycol has been shown to contain substantial amounts of aldehydes, peroxides, iron, and uv-absorbing hydrocarbons. These impurities can be removed by reduction with sodium borohydride, dilution with H2O, passing through a train of four columns, and filtering through a 0.45-micron filter. The product is stable for at least several months and perhaps much longer; storage under nitrogen in acid-washed dark bottles is preferable. Ten liters of 25% (v/v) aqueous ethylene glycol can easily be purified in about 1 week using equipment commonly available in a biochemical laboratory. This purification is also applicable to aqueous glycerol.