The role of the phosphate groups of trinitrophenyl adenosine 5'-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation.

A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Targeting Cancer Metabolism and Current Anti-Cancer Drugs.

Several studies have exploited the metabolic hallmarks that distinguish between normal and cancer cells, aiming at identifying specific targets of anti-cancer drugs. It has become apparent that metabolic flexibility allows cancer cells to survive during high anabolic demand or the depletion of nutrients and oxygen. Cancers can reprogram their metabolism to the microenvironments by increasing aerobic glycolysis to maximize ATP production, increasing glutaminolysis and anabolic pathways to support bioenergetic and biosynthetic demand during rapid proliferation. The increased key regulatory enzymes that support the relevant pathways allow us to design small molecules which can specifically block activities of these enzymes, preventing growth and metastasis of tumors. In this review, we discuss metabolic adaptation in cancers and highlight the crucial metabolic enzymes involved, specifically those involved in aerobic glycolysis, glutaminolysis, de novo fatty acid synthesis, and bioenergetic pathways. Furthermore, we also review the success and the pitfalls of the current anti-cancer drugs which have been applied in pre-clinical and clinical studies.

A Quantitative Method for the Measurement of Protein Histidine Phosphorylation.

The method described in this chapter provides a quantitative means of assaying for protein histidine phosphorylation and thus protein histidine kinase activity, even in the presence of other protein kinases, for example, serine/threonine or tyrosine kinases. The method involves the measurement of 32P, derived from [γ32P]ATP, incorporation into phosphohistidine in a protein substrate. The method makes use of the differential stabilities of phosphohistidine and the common phosphohydroxyamino acids to alkali and acid treatments to measure phosphohistidine incorporation. Phosphoserine and phosphothreonine are depleted by alkali treatment, while phosphohistidine, which is alkali-stable, is removed by acid treatment. Phosphotyrosine is stable to both alkali and acid treatments. The method is filter-based and allows for rapid assay of multiple protein histidine kinase samples, for example, screening for histidine kinase activity, allowing for the calculation of specific activity. In addition, quantitative time-course assays can also be performed to allow for kinetic analysis of histidine kinase activity.

Roles of pyruvate carboxylase in human diseases: from diabetes to cancers and infection.

Pyruvate carboxylase (PC), an anaplerotic enzyme, plays an essential role in various cellular metabolic pathways including gluconeogenesis, de novo fatty acid synthesis, amino acid synthesis, and glucose-induced insulin secretion. Deregulation of PC expression or activity has long been known to be associated with metabolic syndrome in several rodent models. Accumulating data in the past decade clearly showed that deregulation of PC expression is associated with type 2 diabetes in humans, while targeted inhibition of PC expression in a mouse model reduced adiposity and improved insulin sensitivity in diet-induced type 2 diabetes. More recent studies also show that PC is strongly involved in tumorigenesis in several cancers, including breast, non-small cell lung cancer, glioblastoma, renal carcinoma, and gall bladder. Systems metabolomics analysis of these cancers identified pyruvate carboxylation as an essential metabolic hub that feeds carbon skeletons of downstream metabolites of oxaloacetate into the biosynthesis of various cellular components including membrane lipids, nucleotides, amino acids, and the redox control. Inhibition or down-regulation of PC expression in several cancers markedly impairs their growth ex vivo and in vivo, drawing attention to PC as an anti-cancer target. PC has also exhibited a moonlight function by interacting with immune surveillance that can either promote or block viral infection. In certain pathogenic bacteria, PC is essential for infection, replication, and maintenance of their virulence phenotype.

The actions of NME1/NDPK-A and NME2/NDPK-B as protein kinases.

Nucleoside diphosphate kinases (NDPKs) are multifunctional proteins encoded by the nme (non-metastatic cells) genes, also called NM23. NDPKs catalyze the transfer of γ-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high-energy phosphohistidine intermediate. Growing evidence shows that NDPKs, particularly NDPK-B, can additionally act as a protein histidine kinase. Protein kinases and phosphatases that regulate reversible O-phosphorylation of serine, threonine, and tyrosine residues have been studied extensively in many organisms. Interestingly, other phosphoamino acids histidine, lysine, arginine, aspartate, glutamate, and cysteine exist in abundance but remain understudied due to the paucity of suitable methods and antibodies. The N-phosphorylation of histidine by histidine kinases via the two- or multi-component signaling systems is an important mediator in cellular responses in prokaryotes and lower eukaryotes, like yeast, fungi, and plants. However, in vertebrates knowledge of phosphohistidine signaling has lagged far behind and the identity of the protein kinases and protein phosphatases involved is not well established. This article will therefore provide an overview of our current knowledge on protein histidine phosphorylation particularly the role of nm 23 gene products as protein histidine kinases.

Focus on O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine.

The synthesis and chemistry of the lesser-known phosphoamino acids, O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine are described in detail. In addition, where anything at all is known, the biological synthesis, occurrence and functions of these phosphoamino acids are described. Of these phosphoamino acids, only N 1-phosphotryptophan has not been reported to occur in proteins; however, apart from the roles of S-phosphocysteine in the sugar transporter component (EII) and in catalysis by protein phosphotyrosine phosphatase, little is currently known about the biological roles of the phosphoamino acids when they occur as post-translational modifications.

Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity.

Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase.

His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3'-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305.

Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase.

Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (K(a)) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the k(cat) for pyruvate carboxylation. Part of the effects of the mutations was to increase the K(m) for MgATP and the K(a) for activation by free Mg(2+) determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalyzed bicarbonate-dependent ATP cleavage, carboxylation of biotin, and phosphorylation of ADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein (BCCP) domain and thus the binding of biotin at the BC domain active site. On the basis of the kinetic analysis proposed here, it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor l-aspartate was largely unaffected by the mutation of either Arg427 or Arg472.

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA.

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.

Histone H4 histidine phosphorylation: kinases, phosphatases, liver regeneration and cancer.

Phosphorylation of histone H4 on one or both of its two histidine residues has been known to occur in liver cells for nearly 40 years and has been associated with proliferation of hepatocytes during regeneration of the liver following mechanical damage. More recently, large increases in histone H4 histidine kinase activity have been found to occur associated with proliferation and differentiation of liver progenitor cells following chemical damage that prevents hepatocyte proliferation. In addition, it has been shown this histone H4 histidine kinase activity is elevated nearly 100-fold in human foetal liver and several hundredfold in hepatocellular carcinoma tissue compared with normal adult liver. In the present paper, we review what is currently known about histone H4 histidine phosphorylation, the kinase(s) responsible and the phosphatases capable of catalysing its dephosphorylation, and briefly summarize the techniques used to detect and measure the histidine phosphorylation of histone H4 and the corresponding kinase activity.

Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli.

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.

Activation and inhibition of pyruvate carboxylase from Rhizobium etli.

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA.

2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme.

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase.

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli.

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO(2) to form oxaloacetate and complete the reaction.

Detection and analysis of protein histidine phosphorylation.

Protein histidine phosphorylation is well established as an important part of signalling systems in bacteria, fungi and plants and there is growing evidence of its role in mammalian cell biology. Compared to phosphoserine, phosphothreonine and phosphotyrosine, phosphohistidine is relatively labile, especially under the acidic conditions that were developed to analyse protein phosphorylation. In recent years, there has been an increasing impetus to develop specific methods for the analysis of histidine phosphorylation and assay of histidine kinase activity. Most recently attention has focussed on the application of mass spectrometry to this end. This review provides an overview of methods available for the detection and analysis of phosphohistidine in phosphoproteins, with particular emphasis on the application of mass spectrometric techniques.

Structure, mechanism and regulation of pyruvate carboxylase.

PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme.

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the K(m)s for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5'-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.

Analysis of trimethyl carboxyphosphate by gas chromatography-mass spectrometry.

Analysis of trimethyl carboxyphosphate samples by gas chromatography-mass spectrometry, using typical conditions resulted in significant decomposition of the analyte. Optimization of injection conditions, including conditioning of the injection port liner, produced a dramatic increase in observed peak areas and afforded an effective method for detection of trimethyl carboxyphosphate at the <1 microg mL(-1) level.