Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

S-adenosylmethionine: studies on chemical and enzymatic synthesis

Several methods for the chemical and enzymatic synthesis of (-)-S-adenosylmethionine (AdoMet) are described and compared. Studies on the effects of solvents, pH, methylating reagents, and KI on the coupling of sodium homocysteine thiolate and 5'-chloro-5'-deoxyadenosine led to an improved procedure for the synthesis of (+/-)-AdoMet. The use of trimethylsulfonium iodide as a methylating agent under acidic conditions results in a higher content of the desired (-)-epimer than does the use of CH3I. The enzymatic synthesis of (-)-AdoMet using AdoMet synthetase from an over-producing strain of Escherichia coli is demonstrated and the effect of product inhibition on preparative-scale synthesis is illustrated. A new HPLC technique for separation of the epimeric mixture of AdoMet, which allows small-scale preparation of optically pure AdoMet from the enzyme product, has been developed. With this HPLC technique, evidence that (-)-AdoMet is the sole epimer formed by the enzyme has been shown.     

The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments

A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.     

Tunneling of intermediates in enzyme-catalyzed reactions

A fascinating group of enzymes has been shown to possess multiple active sites connected by intramolecular tunnels for the passage of reactive intermediates from the site of production to the site of utilization. In most of the examples studied to date, the binding of substrates at one active site enhances the formation of a reaction intermediate at an adjacent active site. The most common intermediate is ammonia, derived from the hydrolysis of glutamine, but molecular tunnels for the passage of indole, carbon monoxide, acetaldehyde and carbamate have also been identified. The architectural features of these molecular tunnels are quite different from one another, suggesting that they evolved independently.     

Immunohistochemical localization of androgen receptors in female mole rat (Spalax leucodon) tissues

Androgens exert their effects through androgen receptors (AR) in tissues. We investigated the distribution of AR in female mole rat tissues. Tissues were excised, fixed with 10% formalin and embedded in paraffin. Sections were stained after microwave antigen retrieval for immunohistochemistry. Immunostaining of AR immunostaining was detected in the nucleus or cytoplasm of the cells in the cerebral cortex, cerebellum, anterior pituitary, lung, liver, uterus and skin. Granulosa and some thecal cells in the ovary, cardiac muscle cells and adipose cells exhibited a nuclear reaction for AR. In the kidney, labeling of AR was restricted to the cytoplasm of tubule cells. We found that AR could be detected using immunohistochemistry in the nucleus or cytoplasm or both in the presence of androgens.     

Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase

Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococcus faecalis), unadenylylated glutamine synthetase (Escherichia coli), and carbamoyl-phosphate synthetase (E. coli). ³¹P NMR was used to determine that carbamate kinase uses the B isomer of Ado-5′-(2-thioPPP) in the presence of Mg²⁺. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd²⁺ suggesting that the metal ion does not bind to the β-phosphoryl group or that both Mg²⁺ and Cd²⁺ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5′-(1-thioPP) with Mg²⁺ and Cd²⁺. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5′-(2-thioPPP) at both ATP sites with Mg²⁺ (Raushel et al., 1978J. Biol. Chem.253, 6627). Current experiments show that the stereospecificity is reversed by Cd^2? and that both A and B isomers are used when Zn²⁺ is present. With Ado-5′-(1-thioPPP), the B isomer is used with Mg²⁺, the A isomer with Cd²⁺, and both isomers with Zn²⁺. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH₃)₄ATP as a substrate and thus we can only speculate that the Δ chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co³⁺-ATP analogs utilized by hexokinase (E. K. Jaffe, and M. Cohn, 1978Biochemistry17, 652). If the sulfur of the β-phosphoryl of Ado-5′-(2-thioPPP) binds to the metal ion with carbamate kinase, then the Δ chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Glutamine synthetase reacts with the B isomer of both Ado-5′-(2-thioPPP) and Ado-5′-(1-thioPPP) in the presence of Mg²⁺. When Co²⁺ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH₃)₄ATP is not a substrate for glutamine synthetase. Glutamine synthetase is therefore different from the two previously mentioned enzymes in that it used the opposite A ring configuration for the metal-ATP chelate.     

Substrate-induced inactivation of argininosuccinate lyase by monofluorofumarate and difluorofumarate

Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 degrees C, and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 microM and 0.5%. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min-1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.     

Structural diversity within the mononuclear and binuclear active sites of N-acetyl-D-glucosamine-6-phosphate deacetylase

NagA catalyzes the hydrolysis of N-acetyl-d-glucosamine-6-phosphate to d-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-d-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the alpha-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.     

Virtual screening against metalloenzymes for inhibitors and substrates

Molecular docking uses the three-dimensional structure of a receptor to screen databases of small molecules for potential ligands, often based on energetic complementarity. For many docking scoring functions, which calculate nonbonded interactions, metalloenzymes are challenging because of the partial covalent nature of metal-ligand interactions. To investigate how well molecular docking can identify potential ligands of metalloenzymes using a "standard" scoring function, we have docked the MDL Drug Data Report (MDDR), a functionally annotated database of 95,000 small molecules, against the X-ray crystal structures of five metalloenzymes. These enzymes included three zinc proteases, the nickel analogue of an iron enzyme, and a molybdenum metalloenzyme. The ability of the docking program to retrospectively enrich the annotated ligands as high-scoring hits for each enzyme and to calculate proper geometries was evaluated. In all five systems, the annotated ligands within the MDDR were enriched at least 20 times over random. To test the approach prospectively, a sixth target, the zinc beta-lactamase from Bacteroides fragilis, was screened against the fragment-like subset of the ZINC database. We purchased and tested 15 compounds from among the top 50 top-ranked ligands from docking, and found 5 inhibitors with apparent K(i) values less than 120 microM, the best of which was 2 microM. A more ambitious test still was predicting actual substrates for a seventh target, a Zn-dependent phosphotriesterase from Pseudomonas diminuta. Screening the Available Chemicals Directory (ACD) identified 25 thiophosphate esters as potential substrates within the top 100 ranked compounds. Eight of these, all previously uncharacterized for this enzyme, were acquired and tested, and all were confirmed experimentally as substrates. These results suggest that a simple, noncovalent scoring function may be used to identify inhibitors of at least some metalloenzymes.     

The role of thallium-201 and pentavalent dimercaptosuccinic acid for staging cartilaginous tumours

INTRODUCTION: Heterogeneity of cartilage tumours may confound accurate diagnosis and grading resulting in under and over treatment. Improved preoperative assessment of malignancy and grade would be invaluable for developing a rational plan for treatment. We examined correlations between nuclear tracer avidity and malignancy grade in cartilage tumours. METHODS: Between 1996 and 2000, 92 consecutive patients with cartilaginous tumours (50 benign, 42 non-metastatic malignant) underwent nuclear scanning. Thallium-201 (TL-201) and pentavalent dimercaptosuccinic acid (DMSAV) were used as nuclear isotopes. Scanning with these agents was performed on separate days 48 hours apart. Static and SPECT images were obtained at 30 m and 4 h after injection of nuclear tracer. Pathology review was undertaken blinded to the results of the nuclear scans and correlations between histologic results and trace uptake at 4 hours examined. RESULTS: 25 patients with negative DMSAV had benign tumours. 15/17 tumours with positive TL-201 had malignant tumours. 11/13 patients with both positive DMSAV and TL-201 scans had intermediate or high grade tumours and 4 of these developed metastases. We have developed an algorithm for the management of patients with tumours that aims to avoid over treatment of low grade tumours and under treatment of high grade tumours. CONCLUSION: Functional nuclear scanning with TL-201 and DMSAV complements other imaging modalities in the management of cartilaginous tumours.