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The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) kcat and kcat/Km values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol–1 increases in ΔG† for kcat/Km for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol–1 increases, respectively, in ΔG†. The D37G and T100′A substitutions both resulted in smaller 2 kcal mol–1 decreases in ΔG† for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol–1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5’-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5’-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100’ side chains of OMPDC interact, respectively, with the C-3’ and C-2’ hydroxyl groups of enzyme-bound OMP. D37G and T100’A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG(‡) for catalysis of decarboxylation of the phosphodianion truncated substrate 1-(β-D-erythrofuranosyl)orotic acid (EO), but in larger 2.1–2.9 kcal/mole increases in ΔG(‡) for decarboxylation of OMP, and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition state stabilization by the Q215, Y217 and R235 side chain at the dianion binding site. The D37G and T100’A substitutions result in

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 A resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

The movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on k(cat) in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces k(cat) by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.

Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O–O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.

Two imidazole groups act together to catalyze the hydrolysis of the phosphodiester bis(2-(1-methyl-1H-imidazolyl)phenyl) phosphate (BMIPP). A full investigation involving searching computational and electrospray ionization (ESI-MS-/MS) and ultra mass spectrometry (LTQ-FT) experiments made possible a choice between two kinetically equivalent mechanisms. The preferred pathway, involving intramolecular nucleophilic catalysis by imidazole, assisted by intramolecular general acid catalysis by the imidazolium group, offers the first simple model for the mechanism used by the extensive phospholipase D superfamily.

Biochemical reactions of phosphate and sulfate esters are ubiquitous in the living world, and are found throughout many pathways involving metabolism, biosynthesis, control of transcription, energy storage, and replication. This chapter describes the mechanisms by which phosphate and sulfate monoesters undergo uncatalyzed and enzyme-catalyzed hydrolysis by phosphatases and sulfatases, respectively. Relative to the slow rates of the uncatalyzed reactions of their substrates, these enzymes produce rate accelerations that are among the greatest of any enzymes yet described. Interestingly, nature has evolved several quite different catalytic strategies to accomplish this. This review summarizes the current state of knowledge of several phosphatases and sulfatases. Phosphatases and sulfatases constitute a large group of enzymes, with structural and mechanistic diverseness. Within each group, one finds enzymes that utilize completely different catalytic machinery and mechanisms to accomplish the same reaction. We also summarize the mechanistic tools that have been used to study the chemistry and biochemistry of sulfate and phosphate esters, with references to comprehensive treatments of these methods for those who wish more thorough explanations of the tools of enzymology and physical organic chemistry.

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 A structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO](2) core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

Ammonia oxide is revealed as a stable molecule in a crystal structure and as a likely reactive species in many reactions of hydroxylamine.

This work describes the effect of a variety of metal ions as quenchers of the fluorescence of naphthalene, in aqueous micellar solutions of sodium dodecyl sulfate (SDS). The quenching by the metal ions can be adequately described by the Stern–Volmer equation and the best signal to noise ratios are obtained with low micellized detergent concentrations. Apparent Stern–Volmer constants decrease in the order: Fe3+ > Cu2+ > Pb2+ > Cr3+ > Ni2+ and directly reflect the relative sensitivity of the method for these ions. Detection limits (defined as three times the standard deviation of the blank for n = 10) for the fluorescence quenching of naphthalene by the metal ions in aqueous micellar SDS are in the range of 1.0 × 10−6 to 1.0 × 10−5 mol dm−3. The proposed fluorescence quenching method shows good repeatibility for a variety of added quencher metal ions, indicating that anionic micelle-enhanced fluorescence quenching by metal ions constitutes an analytical method of rather general application.

The hydrolysis of the phosphate diester bis(2-(1-methyl-1H-imidazolyl)phenyl) phosphate BMIPP was evaluated as an intramolecular model for the ribonuclease A mechanism. Kinetic and thermodynamic data, isotope effects and ESI-MS and ESI-MS/MS analysis support the proposed intramolecular general acid−base catalysis by the imidazole groups.