Paul V. Attwood

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 This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways.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.

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.

Focus on phosphohistidine

Summary.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 ΔG° 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.

Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme

Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.

Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes.

Biotin-dependent carboxylases catalyze a variety of carboxyl transfer reactions in a number of metabolic pathways and are found in all free-living organisms. They are large molecules which can comprise a single polypeptide chain with three domains or up to three subunits, each of which performs a particular part of the overall reaction. Biotin plays a central role in the action of these enzymes. In this Account we examine the current state of knowledge of the chemistry of catalysis and consider how the recent explosion of available protein sequence and structural information has assisted our understanding of the mechanisms of biotin-dependent enzymes.

Mammalian protein histidine kinases

The existence of protein kinases, known as histidine kinases, which phosphorylate their substrates on histidine residues has been well documented in bacteria and also in lower eukaryotes such as yeast and plants. Their biological roles in cellular signalling pathways within these organisms have also been well characterised. The evidence for the existence of such enzymes in mammalian cells is much less well established and little has been determined about their cellular functions. The aim of the current review is to present a summary of what is known about mammalian histidine kinases. In addition, by consideration of the chemistry of phosphohistidine, what is currently known of some mammalian histidine kinases and the way in which they act in bacteria and other eukaryotes, a general role for mammalian histidine kinases is proposed. A histidine kinase phosphorylates a substrate protein, by virtue of the relatively high free energy of hydrolysis of phosphohistidine the phosphate group is easily transferred to either a small molecule or another protein with which the phosphorylated substrate protein specifically interacts. This allows a signalling process to occur, which may be downregulated by the action of phosphatases. Given the known importance of protein phosphorylation to the regulation of almost all aspects of cellular function, the investigation of the largely unexplored area of histidine phosphorylation in mammalian cells is likely to provide a greater understanding of cellular action and possibly provide a new set of therapeutic drug targets.

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.

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.

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.

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.