Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy), playing an essential role in modulating the cellular Hcy levels and regulating activities of a host of methyltransferases in eukaryotic cells. This enzyme exists in an open conformation (active site unoccupied) and a closed conformation (active site occupied with substrate or inhibitor) [Turner, M. A., Yang, X., Yin, D., Kuczera, K., Borchardt, R. T., and Howell, P. L. (2000) Cell Biochem. Biophys. 33, 101-125]. To investigate the binding of natural substrates during catalysis, the computational docking program AutoDock (with confirming calculations using CHARMM) was used to predict the binding modes of various substrates or inhibitors with the closed and open forms of AdoHcy hydrolase. The results have revealed that the interaction between a substrate and the open form of the enzyme is nonspecific, whereas the binding of the substrate in the closed form is highly specific with the adenine moiety of a substrate as the main recognition factor. Residues Thr57, Glu59, Glu156, Gln181, Lys186, Asp190, Met351, and His35 are involved in substrate binding, which is consistent with the crystal structure. His55 in the docked model appears to participate in the elimination of water from Ado through the interaction with the 5'-OH group of Ado. In the same reaction, Asp131 removes a proton from the 4' position of the substrate after the oxidation-reduction reaction in the enzyme. To identify the residues that bind the Hcy moiety, AdoHcy was docked to the closed form of AdoHcy hydrolase. The Hcy tail is predicted to interact with His55, Cys79, Asn80, Asp131, Asp134, and Leu344 in a strained conformation, which may lower the reaction barrier and enhance the catalysis rate.

In vitro metabolism studies of the prodrug, 2',3',5'-triacetyl-6-azauridine, utilizing an automated analytical system.

The purpose was to study in vitro metabolism of 2',3',5'-triacetyl-6-azauridine (1) by porcine liver esterase (PLE) and in human plasma using an automated analytical system developed previously. A gradient-LC method was developed to study the concentration-time course of 1 and its metabolites. A fast-LC assay was used to study the temperature effect on the metabolism of 1 by the PLE. 1 and all of its proposed possible metabolites were separated by the gradient-LC method in less than 10 min. Two simplified kinetic schemes were developed to describe the time course of 1, the intermediates and final metabolites with only five rate constants for the metabolisms of 1 by PLE and four rate constants in human plasma. Both enthalpy and entropy of activation in the in vitro metabolism of 1 by PLE were obtained.

Formaldehyde production by Tris buffer in peptide formulations at elevated temperature.

This technical note provides evidence for the degradation of Tris buffer in a peptide formulation stored at elevated temperature (70 degrees C). The buffer degrades to liberate formaldehyde, which is shown to react with the peptide tyrosine residue. Those involved in peptide/protein formulation should be aware of the possible instability in this common biological buffer.

A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells.

PURPOSE: To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers. METHODS: A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (K(I) values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. RESULTS: [3H]-Taxol (0.04 microM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (+/-)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting K(I) values of 1.5+/-0.8, 2.5+/-1.0, 3.0+/-0.3, 7.3+/-0.7, 8.5+/-2.8, 36.5+/-1.5, 276+/-69, and 313+/-112 microM, respectively. With the exception of that of quinidine, these K(I) values were comparable with literature values. CONCLUSIONS: This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp, however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

Effect of 'pH' on the rate of asparagine deamidation in polymeric formulations: 'pH'-rate profile.

The rate of Asn deamidation of a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala) was measured as a function of effective pH ('pH') in glassy and rubbery polymeric solids containing poly(vinyl pyrrolidone) (PVP) and in solution controls at 70 degrees C. The reaction exhibited pseudo-first-order kinetics in all samples over a wide 'pH' range (0.5 < 'pH' < 12); the formation of similar products suggests that the reaction mechanism is unaffected by matrix type. Rates of deamidation were comparable for the polymeric and solution samples in the acidic range ('pH' < 4). Solution-state rates were faster than those in polymeric solids at neutral 'pH' (6 < 'pH' < 8), increasing to a > 10,000-fold difference in the basic range ('pH' > 8). Specific base catalysis was observed in solution and in the polymeric solids under neutral conditions (6 < 'pH' < 8). In solution, the reaction exhibited general base catalysis for 'pH' > 8, whereas the reaction was 'pH'-independent in the polymeric solids in this range. The 'pH'-rate profile and supporting buffer catalysis data are consistent with a change in the rate-determining step in the basic range from 'pH'-dependent attack of the deprotonated backbone amide nitrogen on the Asn side chain in solution to 'pH'-independent ammonia expulsion in the polymeric solids. The results suggest that polymer matrix incorporation not only affects the magnitude of the deamidation rate constant but also the 'pH' dependency of the reaction and the rate-determining step in the basic 'pH' range.

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.

Reactivity toward deamidation of asparagine residues in beta-turn structures.

Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn.

Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation.

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.

Catalytic antibodies for complex reactions: hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction.

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.

Hydrogen bonds and proton transfer in general-catalytic transition-state stabilization in enzyme catalysis.

The question of the nature of the proton bridge involved in general acid-base catalysis in both enzymic and non-enzymic systems is considered in the light of long-known but insufficiently appreciated work of Jencks and his coworkers and of more recent results from neutron-diffraction crystallography and NMR spectroscopic studies, as well as results from isotope-effect investigations. These lines of inquiry lead toward the view that the bridging proton, when between electronegative atoms, is in a stable potential at the transition state, not participating strongly in the reaction-coordinate motion. Furthermore they suggest that bond order is well-conserved at unity for bridging protons, and give rough estimates of the degree to which the proton will respond to structural changes in its bonding partners. Thus if a center involved in general-catalytic bridging becomes more basic, the proton is expected to move toward it while maintaining a unit total bond order. For a unit increase in the pK of a bridging partner, the other partner is expected to acquire about 0.06 units of negative charge. The implications are considered for charge distribution in enzymic transition states as the basicity of catalytic residues changes in the course of molecular evolution or during progress along a catalytic pathway.

Deamidation of a model hexapeptide in poly(vinyl alcohol) hydrogels and xerogels.

Polymeric controlled release systems have been proposed to prolong the half-lives of protein and peptide drugs in vivo and to deliver active drug at a controlled rate. These systems are ineffective, however, if the drug is not stable during storage and release. This study addresses the effect of poly(vinyl alcohol) on the stability and release of an incorporated hexapeptide, VYPNGA, which undergoes deamidation. Two types of peptide-loaded poly(vinyl alcohol) matrices were formed, a semisolid hydrogel and a lower water content 'xerogel', and stored at 50 degrees C for up to 122 days. The hexapeptide was less stable in both poly(vinyl alcohol) matrices than in aqueous buffer or lyophilized polymer-free powders. The type of poly(vinyl alcohol) matrix appeared to influence the degradation mechanism, since the product distributions differ in the hydrogel and the xerogel. The results suggest that, rather than stabilizing this peptide, incorporation in poly(vinyl alcohol) matrices reduces stability relative to solution and lyophilized controls.

Chemical stability of peptides in polymers. 1. Effect of water on peptide deamidation in poly(vinyl alcohol) and poly(vinyl pyrrolidone) matrixes.

This paper examines the effect of water content, water activity, and glass transition temperature (T(g)) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 degrees C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing T(g). The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or T(g)) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of T(g) from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 degrees C, suggesting that the physical state of formulations could be used to predict chemical stability.

Chemical stability of peptides in polymers. 2. Discriminating between solvent and plasticizing effects of water on peptide deamidation in poly(vinylpyrrolidone).

The mechanistic role of water in the deamidation of a model asparagine-containing hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations containing poly(vinylpyrrolidone) (PVP) and glycerol was investigated. Glycerol was used as a plasticizer to vary formulation glass transition temperature (T(g)) without significantly changing water content or activity. Increases in moisture and glycerol contents increased the rate of peptide deamidation. This increase was strongly correlated with T(g) at constant water content and activity, suggesting that increased matrix mobility facilitates deamidation. In rubbery systems (T > T(g)), deamidation rates appeared to be independent of water content and activity in formulations with similar T(g)s. However, in glassy formulations with similar T(g)s, deamidation increased with water content, suggesting a solvent/medium effect of water on reactivity in this regime. An increase in water content also affected the degradation product distribution; less of the cyclic imide intermediate and more of the hydrolytic products, isoAsp- and Asp-hexapeptides, were observed as water content increased. Thus, residual water appears to facilitate deamidation in these solid PVP formulations both by enhancing molecular mobility and by solvent/medium effects, and also participates as a chemical reactant in the subsequent breakdown of the cyclic imide.

The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase.

The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P. Meth. Enzymol. 1977, 46, 48). This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde. Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site. The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.

Secondary structure and protein deamidation.

The deamidation reactions of asparagine residues in alpha-helical and beta-turn secondary structural environments of peptides and proteins are reviewed. Both kinds of secondary structure tend to stabilize asparagine residues against deamidation, although the effects are not large. The effect of beta-sheet structures on asparagine stability is unclear, although simple considerations suggest a stabilization in this environment also.

Transition-state theoretical interpretation of the catalytic power of pyruvate decarboxylases: the roles of static and dynamical considerations.

The catalytic power of two thiamin diphosphate (ThDP)-dependent enzymes, yeast pyruvate decarboxylase (the hysteretically regulated enzyme from Saccharomyces cerevisiae, SCPDC) and bacterial pyruvate decarboxylase (the unregulated enzyme from Zymomonas mobilis, ZMPDC), are analyzed by thorough-going application of transition-state theory, i.e. by a static approach that emphasizes the state-function character of the free energy of activation and takes no explicit account of dynamical considerations. The overall catalytic reaction is resolved into manifolds for addition (conversion of free enzyme and substrate to the complex of enzyme with the pyruvate:ThDP adduct), decarboxylation, and elimination (conversion of the complex of enzyme with the acetaldehyde:ThDP adduct formed by decarboxylation into free product and free enzyme). For SCPDC, the addition manifold is most strongly catalyzed (3x1012-fold, corresponding to net transition-state stabilization of 72 kJ/mol, transition-state stabilization of 83 kJ/mol diminished by reactant-state stabilization of 11 kJ/mol), the decarboxylation manifold is least strongly catalyzed (5x107-fold, corresponding to net transition-state stabilization of 41 kJ/mol, transition-state stabilization of 68 kJ/mol diminished by reactant-state stabilization of 27 kJ/mol), and the elimination manifold is catalyzed to an intermediate degree (2x1010-fold, corresponding to net transition-state stabilization of 59 kJ/mol, transition-state stabilization of 76 kJ/mol diminished by reactant-state stabilization of 17 kJ/mol). A similar situation holds for ZMPDC. There is no need to make an explicit analysis of dynamical factors in order to describe the catalytic mechanism and catalytic power of these complex enzymes.

Ion channel properties of a protein complex with characteristics of a glutamate/N-methyl-D-aspartate receptor.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Automated analytical systems for drug development studies. V. A system for enzyme kinetic studies.

Two similar automated analytical systems using liquid chromatography (LC) and microdialysis as an on-line sampling technique were applied to studies of enzyme kinetics. 2',3',5'-Triacetyl-6-azauridine (azaribine) with porcine liver esterase (PLE) and N-acetylphenylalanyl-3,5-diiodotyrosine (AcFY') with pepsin were used as model compounds. The microdialysis sampling technique permitted the rapid separation of low molecular weight analytes from macromolecules, thus simultaneously achieving clean-up of the samples and quenching of the reaction. The combination of rapid LC analysis and microdialysis sampling provided selectivity and automation. The systems are rugged and give reproducible results in agreement with those from manual sampling methods.