James K. Coward

Use of aminopropyltransferase inhibitors and of non-metabolizable analogs to study polyamine regulation and function.

The polyamines spermidine and spermine are essential for the growth of mammalian cells. This review describes the properties of the two aminopropyltransferases that are responsible for their biosynthesis, the synthesis and use of specific aminopropyltransferase inhibitors, and the use of analogs of the polyamines to investigate polyamine transport and function. Highly specific and potent multisubstrate adduct inhibitors of these enzymes have been synthesized while less potent inhibitors have been obtained by the synthesis of amines that bind at the active site. Studies with these inhibitors indicate that polyamines are needed for a normal rate of growth and that, although some of the functions of polyamines may be interchangeable, other functions may have a specific requirement for spermidine or spermine. Two groups of growth-promoting polyamine analogs can be distinguished: the many that are effective in short-term experiments compared to the few that can act over a prolonged period. The more stringent structural requirements for long-term growth are probably due to a need for spermidine, or a closely related analog, as a precursor of hypusine in the protein eIF-5A. Metabolically resistant polyamine analogs can be used as model substrates for studies of the polyamine transport system, which plays a critical role in maintaining normal cellular polyamine levels. The feedback regulation by high levels of polyamines that downregulates transport is essential to prevent the accumulation of polyamines at toxic levels. Such accumulation may be associated with apoptosis and, therefore, polyamine analogs are useful tools for investigating the mechanism(s) of polyamine-mediated toxicity.

Specific and potent inhibition of spermidine synthase by the transition-state analog, S-adenosyl-3-thio-1,8-diaminooctane

Abstract The title compound ( 1 c ), was designed and synthesized based on mechanistic data concerning enzyme-catalyzed alkyl transfer reactions, applied in this case to aminopropyl transferases. The inhibition by 1 c of one such enzyme, spermidine synthase, was both potent (I 50 = 4 × 10 −7 M) and specific. A closely related aminopropyltransferase, spermine synthase was only minimally affected by high concentrations of 1 c . Similar, although not as marked, specifity between the two aminopropyltransferases was observed with the corresponding methyl sulfonium salt, 2 c . Studies with structurally related compounds support the hypothesis that the strong inhibition of spermidine synthase by 1 c derives from the incorporation in this compound of important features of the transition-state structure of this enzyme-catalyzed reaction.

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP‐dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis—amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C‐terminal of GspS belongs to the ATP‐grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH‐binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

A continuous spectrophotometric assay for catechol-O-methyltransferase

Abstract Catechol-O-methyl transferase (COMT) activity can be monitored continuously using a coupled enzyme assay in which the inhibitory product S-adenosylhomocysteine (SAH) is converted to S-inosylhomocysteine (SIH). A simple spectrophotometric assay for COMT is described based on the difference in the ultraviolet absorption spectra between SAH and SIH.

Separation of substituted pteroyl monoglutamates and pteroyl oligo-γ-l-glutamates by high pressure liquid chromatography

Abstract Rapid analysis of substituted and unsubstituted pteroyl-oligo-γ- l -glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.

Dissection of Glutathionylspermidine Synthetase/Amidase from Escherichia coli into Autonomously Folding and Functional Synthetase and Amidase Domains

The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1,4-diaminobutane) and the glycine carboxylate of glutathione (γ-Glu-Cys-Gly) and the opposing hydrolysis of this amide bond (Bollinger, J. M., Jr., Kwon, D. S., Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) J. Biol. Chem. 270, 14031-14041). In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains. In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules. A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein. A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs. This observation suggests that the amidase activity in the full-length protein is negatively autoregulated. The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage. No metal ion is required for the amidase activity. A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki ∼ 10 μM) but does not inhibit the amidase activity.

Phosphonate and phosphinate analogues of N-acylated γ-glutamylglutamate: Potent inhibitors of glutamate carboxypeptidase II

Phosphonate and phosphinate analogues of N-acylated gamma-glutamylglutamate were tested for the ability to inhibit glutamate carboxypeptidase II (GCP II). All of the compounds inhibit GCP II with IC(50) values in the low nanomolar range. The comparison of the results to previously reported inhibitory studies of the same compounds toward folylpoly-gamma-glutamyl synthetase (FPGS) and gamma-glutamyl hydrolase (gamma-GH) provides insight into structural and mechanistic features of each enzyme. Potential utility of these compounds as diagnostic agents and probes to understand folate or antifolate poly-gamma-glutamates metabolism is also described.

Retention behavior of pteroyl-oligo-γ-l-glutamates in reversed-phase chromatography

The effect of eluent pH on the retention of pteroyl-oligo-γ-l-glutamates containing up to eight glutamyl residues is investigated in reversed-phase chromatography with octadecyl-silica column. When the carboxylic groups of the solutes are largely undissociated, at pH 2, the retention of oligoglutamates increases with the number of glutamyl residues and the elution order parallels that in anion-exchange chromatography. At sufficiently high eluent pH the carboxylic groups are dissociated and the elution order is reversed so that solute molecules having smaller number of charges. i.e. less glutamyl residues, are retained stronger. The logarithm of capacity factor, with the exception of folic acid, is linearly dependent on the number of glutamyl residues over a wide range of eluent pH. The dependence of the capacity factor on pH for oligoglutamates is quantitatively interpreted considering the different dissociation constants for the α- and γ-carboxyls. The results suggest that in reversed-phase chromatography the selectivity of separation for polyionogenic compounds can be drastically modulated by changing the pH of the eluent.

Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine

Bacterial aminopropyltransferases from Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa were strongly inhibited by S‐adenosyl‐1,8‐diamino‐3‐thiooctane (AdoDATO) and by dicyclohexylamine. The sensitivity to these drugs in vitro was comparable to that of mammalian spermidine synthase, but AdoDATO was much less potent in reducing spermidine content in the bacteria than in mammalian cells. Although AdoDATO was a stronger inhibitor than dicyclohexylamine in vitro, dicyclohexylamine was more active in reducing bacterial spermidine levels in vivo, suggesting that it is take up better or is more stable in the cell and is the preferable compound for in vivo studies in microorganisms. The strong inhibition of spermidine synthases by AdoDATO which is a transition state analog supports the concept that these enzymes proceed by a single displacement reaction, rather than by a ping‐pong mechanism.

UDP-(5F)-GlcNAc Acts as a Slow-Binding Inhibitor of MshA, a Retaining Glycosyltransferase

Glycosyltransferase enzymes play important roles in numerous cellular pathways. Despite their participation in many therapeutically relevant pathways, there is a paucity of information on how to effectively inhibit this class of enzymes. Here we report that UDP-(5F)-GlcNAc acts as a slow-binding, competitive inhibitor of the retaining glycosyltransferase MshA from Corynebacterium glutamicum (K(i) approximately 1.6 muM). The kinetic data are consistent with a single-step inhibition mechanism whose equilibration is slow relative to catalysis. We believe that this is the first slow-onset inhibitor to be reported for the glycosyltransferase family of enzymes. The potent inhibition of the enzyme by the fluoro-substituted substrate is consistent with the involvement of an oxocarbenium transition-state structure, which has been previously proposed for this family of enzymes. Additionally, although several members of the GT-B enzyme family, including MshA, have been shown to undergo a conformational change upon UDP-GlcNAc binding, the kinetic data are inconsistent with a two-step inhibition mechanism. This suggests that there may be other conformations of the enzyme that are useful for the design of inhibitors against the large family of GT-B glycosyltransferase enzymes.