Transition-state Acidities and the pH Dependence of Drug Stability

The pH dependence of hydrolytic reactions of drugs allows some control of their stability through adjustment of the pH of storage. The transition-state acidity concept of J. L. Kurz is shown to apply to the systematics of relevant pH rate profiles. As limiting cases for the hydrolysis of a series of carboxylic acid derivatives (often employed in pro-drug modifications) in neutral and basic solution, two situations are considered: 1) larger structural effects in basic solution, in which case the most stable compound of the series has the highest transition-state pKa, and 2) larger structural effects in neutral solution, in which case the most stable compound will have the lowest transition-state pKa. The former is the expected situation for variations of reactant electronic features because the negatively charged transition state for the hydroxide-promoted reaction in basic solution should respond more sensitively to electronic effects than should the dipolar transition state for the »un- catalyzed« reaction in neutral solution. Available data for some important substrates in fact do not show the expected behavior, which may be indicative of a concerted reaction (no tetrahedral intermediate) for reactive substrates with hydroxide ion, a mechanism for which others have already provided evidence, and possible reaction through an ion pair for the reaction of reactive substrates with water.

Isotope Effects and Temperature Dependences in the Action of the Glucose Dehydrogenase of the Mesophilic Bacterium Bacillus megaterium

This is the peer reviewed version of the following article: Anandarajah K., Schowen K. B. and Schowen R. L. (2013), Isotope effects and temperature dependences in the action of the glucose dehydrogenase of the mesophilic bacterium Bacillus megaterium, Journal of Physical Organic Chemistry. doi: 10.1002/poc.3166, which has been published in final form at http://doi.org/10.1002/poc.3166. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.The glucose dehydrogenase of the mesophilic bacterium Bacillus megaterium (optimal growth around 35 °C) exhibits non-linear Eyring temperature dependences from 25 to 55 °C in its catalysis of the oxidation by hydride-transfer to NAD+ of the β-anomers of 1-h-D-glucose and 1-d-D-glucose (rate constant kcat/KMβ). A break around 300K separates a high-T region from a low-T region. In the high-T region, isotopic enthalpies of activation within a considerable experimental error are equal to zero. In the low-T region, the enthalpies of activation are roughly equal for the isotopic substrates but are different from zero. An alternative treatment with Eyring plots taken as effectively linear produces enthalpies of activation having the unusual feature of being larger for the H-substrate (26 kJ/mol) than for the D-substrate (21 kJ/mol). Compensation of the enthalpic effect by a more positive entropy for the H-substrate then reproduces the isotope effects. For oxidation by NADP+ of the same pair of isotopic glucose substrates, catalysis by the glucose dehydrogenase of Thermoplasma acidophilum, a thermophilic archaeon, leads to temperature dependences characterized by a high-T region and a low-T region separated by a gentle thermal transition (K. Anandarajah, K.B. Schowen, and R.L. Schowen, Z. phys. Chem. 2008, 222, 1333–1347). Tentative approaches to a mechanistic interpretation of both cases rely on models featuring configurational searches of the enzyme for tunneling states, followed by hydrogen-transfer tunneling, although explanations can be constructed also on the basis of simple transition-state stabilization without tunnelling

Comparative Kinetics of Cofactor Association and Dissociation for the Human and Trypanosomal S-Adenosylhomocysteine Hydrolases. 3. Role of Lysyl and Tyrosyl Residues of the C-Terminal Extension

Based on the available X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs), free energy simulations employing the MM-GBSA approach were applied to predict residues important to the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within 5 Å of the cofactor NAD+/NADH binding site. Among the 38 residues in this region, only four are different between the two enzymes. Surprisingly, the four non-identical residues make no major contribution to differential cofactor binding between Hs-SAHH and Tc-SAHH. On the other hand, four pairs of identical residues are shown by free energy simulations to differentiate cofactor binding between Hs-SAHH and Tc-SAHH. Experimental mutagenesis was performed to test these predictions for a lysine residue and a tyrosine residue of the C-terminal extension that penetrates a partner subunit to form part of the cofactor binding site. The K431A mutant of Tc-SAHH (TcK431A) loses its cofactor binding affinity but retains the wild type’s tetrameric structure, while the corresponding mutant of Hs-SAHH (HsK426A) loses both cofactor affinity and tetrameric structure (Ault-Riche et al., 1994 J Biol Chem, 269, 31472–8). The tyrosine mutants HsY430A and TcY435A alter the NAD+ association and dissociation kinetics, with HsY430A increasing the cofactor equilibrium dissociation constant from approximately 10 nM (Hs-SAHH) to about 800 nM while TcY435A increases the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to about 1 mM. Both changes result from larger increases in off-rate combined with smaller decreases in on-rate. These investigations demonstrate that computational free energy decomposition may be used to guide experimental studies by suggesting sensitive sites for mutagenesis. Our finding that identical residues in two orthologous proteins may give significantly different binding free energy contributions strongly suggests that comparative studies of homologous proteins should investigate not only different residues, but also identical residues in these proteins

Evaluation of NAD(H) analogues as selective inhibitors for Trypanosoma cruzi S-Adenosylhomocysteine hydrolase

This is an Accepted Manuscript of an article published by Taylor & Francis in Nucleosides, Nucleotides and Nucleic Acids in May 2009, available online: http://www.tandfonline.com/10.1080/15257770903044572.S-Adenosylhomocysteine (AdoHcy) hydrolases (SAHHs) from human sources (Hs-SAHHs) bind the cofactor NAD+ more tightly than several parasitic SAHHs by around 1000-fold. This property suggests the cofactor binding site of this essential enzyme as a potential anti-parasitic drug target, e.g., against SAHH from Trypansoma cruzi (Tc-SAHH). The on-rate and off-rate constants and the equilibrium dissociation constants were determined for NAD+/NADH analogues and suggested that NADH analogues were the most promising for selective inhibition of Tc-SAHH. None significantly inhibited Hs-SAHH while S-NADH and H-NADH (Fig. 1) reduced the catalytic activity of Tc-SAHH to <10% in six minutes of exposure

The Rationale for Targeting the NAD/NADH Cofactor Binding Site of Parasitic S-Adenosyl-L-homocysteine Hydrolase for the Design of Anti-parasitic Drugs

This is an Accepted Manuscript of an article published by Taylor & Francis in Nucleosides, Nucleotides and Nucleic Acids in May 2009, available online: http://www.tandfonline.com/10.1080/15257770903051031.Trypanosomal S-adenoyl-L-homocysteine hydrolase (Tc-SAHH), considered as a target for treatment of Chagas disease, has the same catalytic mechanism as human SAHH (Hs-SAHH) and both enzymes have very similar X-ray structures. Efforts toward the design of selective inhibitors against Tc-SAHH targeting the substrate binding site have not to date shown any significant promise. Systematic kinetic and thermodynamic studies on association and dissociation of cofactor NAD/H for Tc-SAHH and Hs-SAHH provide a rationale for the design of anti-parasitic drugs directed toward cofactor-binding sites. Analogues of NAD and their reduced forms show significant selective inactivation of Tc-SAHH, confirming that this design approach is rational

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS

Transition-state vibrational analysis and isotope effects for COMT-catalyzed methyl transfer

Isotopic partition-function ratios (IPFRs) computed for transition structures (TSs) of the methyl-transfer reaction catalyzed by catechol O-methyltransferase and modeled by hybrid QM/MM methods are analyzed. The ability of smaller Hessians to reproduce trends in α-3H3 and 14Cα IPFRs as obtained using the much larger subset QM/MM Hessians from which they are extracted is investigated critically. A 6-atom-extracted Hessian reproduces perfectly the α-T3 IPFR values from the full-subset Hessians of all the TSs but not the α-14CIPFRs. Average AM1/OPLS-AA harmonic frequencies and mean-square amplitudes are presented for the 12 normal modes of the α-CH3 moiety within the active site of several enzymic transition structures, together with QM/MM potential energy scans along each of these modes to assess the degree of anharmonicity. A novel investigation of ponderal effects upon IPFRs suggests that the value for α-14C tends toward a limiting minimum whereas that for α-T3 tends toward a limiting maximum as the mass of the rest of the system increases. The transition vector is dominated by motions of atoms within the donor and acceptor moieties and is very well described as a simple combination of Walden-inversion “umbrella” bending and asymmetric stretching of the SCα and CαO bonds. The contribution of atoms of the protein residues Met40, Tyr68, and Asp141 to the transition vector is extremely small. Average valence force constants for the COMT TS show significant differences from early BEBOVIB estimates which were used in support of the compression hypothesis for catalysis. There is no correlation between TS IPFRs and the nonbonded distances for close contacts between the S atom of SAM and Tyr68 or between any of the H atoms of the transferring methyl group and either Met40 or Asp141