Focus on phosphoaspartate and phosphoglutamate.

Protein phosphorylation is a common signalling mechanism in both prokaryotic and eukaryotic organisms. Whilst the focus of protein phosphorylation research has primarily been on protein serine/threonine or tyrosine phosphorylation, there are other phosphoamino acids that are also biologically important. Two of the phosphoamino acids that are functionally involved in the biochemistry of protein phosphorylation and signalling pathways are phosphoaspartate and phosphoglutamate, and this review focuses on their chemistry and biochemistry. In particular, we cover the biological aspects of phosphoaspartate and phosphoglutamate in signalling pathways and as phosphoenzyme intermediates. In addition, we examine the synthesis of both of these phosphoamino acids and the chemistry of the acyl phosphate group. Although phosphoaspartate is a major component of prokaryotic two-component signalling pathways, this review casts its net wider to include reports of phosphoaspartate in eukaryotic cells. Reports of phosphoglutamate, although limited, appear to be more common as free phosphoglutamate than those found in phosphoprotein form.

Focus on phosphoarginine and phospholysine.

Protein phosphorylation is a common signaling mechanism in both prokaryotic and eukaryotic organisms. Whilst serine, threonine and tyrosine phosphorylation dominate much of the literature there are several other amino acids that are phosphorylated in a variety of organisms. Two of these phosphoamino acids are phosphoarginine and phospholysine. This review will focus on the chemistry and biochemistry of both phosphoarginine and phospholysine. In particular we focus on the biological aspects of phosphoarginine as a means of storing and using metabolic energy (in place of phosphocreatine in invertebrates), the chemistry behind its synthesis and we examine the chemistry behind its highenergy phosphoramidate bond. In addition we will be reporting on the incidence of phosphoarginine in mammalian cells. Similarly we will be reviewing the current findings on the biology and the chemistry of phospholysine and its involvement in a variety of biological systems.

Mass spectrometric analysis of protein histidine phosphorylation.

Protein histidine phosphorylation is now recognized as an important form of post-translational modification. The acid-lability of phosphohistidine has meant that this phosphorylation has not been as well studied as serine/threonine or tyrosine phosphorylation. We show that phosphohistidine and phosphohistidine-containing phosphopeptides derived from proteolytic digestion of phosphohistone H4 are detectable by ESI-MS. We also demonstrate reverse-phase HPLC separation of these phosphopeptides and their detection by MALDI-TOF-MS.

Focus on phosphohistidine.

Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P-N) bond of phosphohistidine has a high DeltaG degrees of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies. Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.

Effects of Mg(2+) on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase.

The effects of Mg(2+) concentration on the kinetics of both ATP cleavage and carboxyenzyme formation in the approach to steady state of the biotin carboxylation reaction of pyruvate carboxylase have been studied. It was found that the enzyme underwent dilution inactivation at low Mg(2+) concentrations and that this occurred at higher enzyme concentrations than had been previously observed. At 10 mM Mg(2+), dilution inactivation was prevented and activation of the enzyme also occurred. When the enzyme was mixed with an ATP solution to initiate the carboxylation reaction, dilution inactivation was reversed and further enzyme activation was induced to a final level that was dependent on Mg(2+) concentration. With the exception of the reaction at 10 mM Mg(2+) in the presence of acetyl CoA, the experimental data could be adequately described as first-order exponential approaches to steady state. At 10 mM Mg(2+) in the presence of acetyl CoA, both ATP cleavage and carboxyenzyme formation data were best described as a biexponential process, in which there was little ATP turnover at steady state. Modeling studies have been performed which produced simulated data that were similar to the experimental data, using a reaction scheme modified from one proposed previously [Legge, G. B., et al. (1996) Biochemistry 35, 3849-3856]. These studies indicate that the major foci of action of Mg(2+) are in the decarboxylation of the enzyme-carboxybiotin complex, the return of the biotin to the site of the biotin carboxylation reaction, and the coupling of ATP cleavage to biotin carboxylation.

Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity.

A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue. This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes. Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source. By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus. The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation. Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine. In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase.

Problems with phosphoamino acid analysis using alkaline hydrolysis.

Although a seemingly simple concept, sample volume and the reaction vessel size are important considerations when undertaking an alkaline hydrolysis of a phosphoprotein, particularly if the phosphoamino acid of interest is either phosphohistidine or phosphotyrosine. It should be noted that the experiments conducted in this article used large concentrations of both phosphotyrosine and the most stable form of phosphohistidine (8), which highlights the problems that may be faced during phosphoamino acid analysis of a protein sample that contains only small amounts of either phosphoamino acid. Although not tested, it is likely that similar hydrolysis effects may occur for phospholysine. If the reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane blot or in solution, then the use of a mineral oil overlay should be considered to prevent concentration of the alkali and hydrolysis of the phosphate moiety.

A chromatographic method for the preparative separation of phosphohistidines.

The driving force behind this new chromatographic technique was to develop a method for purifying preparative quantities of phosphohistidines in a single step that provided good resolution wit eluants that could be easily removed. The current method can provide milligram quantities of each phosphohistidine; however, the later 1H NMR analysis of the dried, individually purified phosphohistidines showed that histidine was present along with each of the individual phosphohistidines. The stability of each phosphohistidine during storage does not appear to be a problem because the amounts of histidine contamination of freshly freeze-dried samples were compared with those of samples stored at -80 degrees C under nitrogen for 2 weeks and were found to be similar (data not shown). Possibly, the lyophilization and preparation of solutions for NMR analysis resulted in a certain amount of hydrolysis of phosphohistidine, particularly with the less stable 1- and 1,3-forms (5, 6). It was noted that when the lyophilized samples were initially dissolved in D2O for NMR analysis, the pH was between 6 and 7; this may have resulted in some hydrolysis of the phosphohistidines. This hydrolysis can be reduced by the addition of 50 mM potassium hydroxide to the pooled fractions collected from the chromatography, so that the alkalinity of the samples is maintained throughout the subsequent processes. The data obtained for the assignment of individual phosphohistidines by 1H and 31P NMR analysis seem consistent with those obtained by other independent studies (6, 10). The NMR analysis was a powerful tool in assigning the identity of each purified phosphohistidine, although caution should be used when considering free phosphohistidines as references for NMR detection of phosphohistidines in native proteins. Lecroisey et al. (10) showed that there were differences between the chemical shifts observed for free phosphohistidine compared to those for phosphohistidine in dipeptides. However, for the purposes of phosphoamino acid analysis, these purified phosphohistidines are used by this group as references in the detection of phosphohistidine in proteins.

Effects of urea on M4-lactate dehydrogenase from elasmobranchs and urea-accumulating Australian desert frogs.

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/K(m(prr)) and V/K(m(NADH)) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the K(m(prr)) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

Effects of acetyl CoA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase.

The approach to steady-state for the formation of the enzyme-carboxybiotin complex obeys first-order kinetics, with the proportion of the total enzyme present as the enzyme-carboxybiotin complex in the steady-state being about 60%. The approach to steady-state for ATP cleavage also obeys first-order kinetics. The apparent first-order rate constants for the approach to steady-state, in the presence and absence of acetyl CoA, respectively, are 6.6 and 0.028 s(-1) for ATP cleavage and 6.1 and 0.028 s(-1) for enzyme-carboxybiotin formation. The similarities of the values of the rate constants for the two reactions indicates that there is a common rate-limiting step. The large enhancement of these rate constants in the presence of acetyl CoA suggests that a major effect of acetyl CoA in the reaction is to enhance the rate of the step in which the putative carboxyphosphate complex is formed and in which ATP is cleaved. In addition, in the presence of acetyl CoA, the formation of the enzyme-carboxybiotin complex is much more tightly coupled to ATP cleavage in the presence of acetyl CoA than in its absence. Modeling studies were performed, and reaction schemes are proposed which give simulations similar to the experimental data. In the reaction schemes, the carboxyphosphate intermediate is able to undergo abortive decomposition without carboxylating biotin. The rate of this abortive reaction is greatly reduced in the presence of acetyl CoA.

Kinetics of nucleotide binding to pyruvate carboxylase.

The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure and the mechanism of action of pyruvate carboxylase.

Pyruvate carboxylase plays an important role in intermediary metabolism, catalysing the formation of oxaloacetate from pyruvate and HCO3-, with concomitant ATP cleavage. It thus provides oxaloacetate for gluconeogenesis and replenishing tricarboxylic acid cycle intermediates for fatty acid, amino acid and neurotransmitter synthesis. The enzyme is highly conserved and is found in a great variety of organisms including fungi, bacteria and plants as well as higher organisms. It is a member of a group of biotin-dependent enzymes and the biotin prosthetic group is covalently bound to the polypeptide chain of the enzyme, there normally being four such chains in the native, tetrameric enzyme. The overall reaction catalysed by pyruvate carboxylase involves two partial reactions that occur at spatially separate subsites within the active site, with the covalently bound biotin acting as a mobile carboxyl group carrier. In the first partial reaction, biotin is carboxylated using ATP and HCO3- as substrates whilst in the second partial reaction, the carboxyl group from carboxybiotin is transferred to pyruvate. The chemical mechanisms of the partial reactions and some of the roles played by amino acid residues of the enzyme in catalysing the reaction have been elucidated. The domain structure of the yeast enzyme has been deduced by comparing its amino acid sequence with those of enzymes that have similar catalytic functions. The quaternary structures of the pyruvate carboxylases studied so far, all involve a tetrahedron-like arrangement of the subunits. The major regulator of enzyme activity, acetyl CoA, stimulates the cleavage of ATP in the first partial reaction and in addition it has been shown to induce a conformational change in the tetrameric structure of the enzyme. In the past, the lack of any detailed structural information on the enzyme has hampered efforts to fully understand how this and other biotin-dependent enzymes function and are regulated. With the recent cloning of the enzyme from a variety of sources and the performance of three-dimensional structural studies, the next few years should see much progress in our understanding the mechanism of action of this enzyme.

Locus of action of acetyl CoA in the biotin-carboxylation reaction of pyruvate carboxylase.

The [14C]carboxyphospho-enzyme complex formed by incubation of the enzyme with H14CO3-, MgATP, and Mg2+ was prepared and isolated by gel filtration as described by Phillips et al. [(1992) Biochemistry 31, 9445-9450]. When time courses of transfer of the [14C]carboxyl group from the complex to pyruvate were studied, it was found that at the first time point (15 s) the formation of [14C]oxalacetate was the same in the presence or absence of acetyl CoA. However, in the absence of acetyl CoA, the radioactivity fixed in [14C]oxalacetate declined rapidly over the subsequent 15 min, whereas in the presence of acetyl CoA the formation of [14C]oxalacetate continued up to about 10 min. The decline in [14C]oxalacetate in the absence of acetyl CoA was found to be due to enzyme-dependent decarboxylation of the oxalacetate by the enzyme. Incubation of the isolated [14C]carboxyphospho-enzyme complex with MgADP and Mg2+ resulted in no significant reduction in the formation of [14C]oxalacetate on addition of acetyl CoA and pyruvate. Incubation of the isolated [32P]carboxyphospho-enzyme complex with pyruvate resulted in no significant reduction in the formation of [gamma-32P]ATP on the addition of MgADP and Mg2+. This new evidence casts doubt on the suggested locus of activation of the enzyme by acetyl CoA being the facilitation of the transfer of the carboxyl group from carboxyphosphate to biotin and indeed on the identity of the isolated enzyme intermediate [Phillips et al. (1992) Biochemistry 31, 9445-9450].

The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation.

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

Characterization of an SHV-5 related extended broad-spectrum beta-lactamase in Enterobacteriaceae from Western Australia.

Among 209 isolates of aminoglycoside resistant Enterobacteriaceae isolated from patients attending the Sir Charles Gairdner Hospital in Perth, Western Australia between January 1985 and June 1991, 41 strains demonstrated additional resistance to cefotaxime, ceftriaxone, ceftazidime and aztreonam. Significant synergy was demonstrable between these beta-lactams and clavulanic acid against resistant strains. The beta-lactamase was mediated by a 170 Kb self-transferring plasmid and identified as the SHV-5 type in isoelectric focusing (pl 8.2) and substrate hydrolysis kinetic studies. The findings record the introduction of extended broad-spectrum beta-lactamases to Australia.