Hughes Goldie

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

SCHEME 1 This conversion is the first committed step of gluconeogenesis in Escherichia coli and is part of the gluconeogenic pathway in virtually all organisms. In bacteria, such as E. coli, PCK is utilized during gluconeogenic growth when sugar levels are low (1). PCK is also an important enzyme in the glycolytic pathways of some organisms, such as Ascaris suum (2) and Trypanosoma cruzi (3), where it forms OAA from PEP, which in turn enters the citric acid cycle. In humans and other mammals, PCK is a central enzyme in carbohydrate metabolism, helping to regulate the blood glucose level. Gluconeogenic tissues, such as kidney and liver, convert lactate and other non-carbohydrate molecules to glucose, which in turn is released into the blood. The importance of PCK to carbohydrate metabolism in humans is such that it has been suggested as a potential drug target in the treatment of non-insulindependent diabetes mellitus (4). PCKs have been traditionally classified according to nucleotide specificity, with the ATP-dependent enzymes found in bacteria, yeast, Trypanomastid parasites and plants, and GTP-dependent PCKs in a variety of other eukaryotes and mammals (5). There are both important differences and similarities between ATPand GTPdependent PCKs. With the exception of bacterial PCKs, which are monomeric (6), most enzymes of the ATP-dependent class are multimeric, with two (7), four (8, 9), or six (10) subunits per enzyme, while known members of the GTP-dependent class are exclusively monomeric. While enzymes of either the ATPor GTP-dependent classes show significant (40–80%) amino acid sequence identity within their respective groups (11, 12), there is no significant overall sequence homology between the two classes of enzyme. Despite this lack of overall homology, both groups of PCKs contain similar NTP and oxaloacetate binding “consensus motifs” in their active sites, which likely play similar roles in substrate binding (which will be described in this review). Also, both GTP-dependent and ATP-dependent PCKs have been shown to possess lysinyl (13–17), argininyl (18, 19), and histidinyl (20, 21) residues at or near their active sites. The differences in nucleotide specificity and kinetic properties between ATPand GTP-dependent PCKs have led to the suggestion that they may be potential therapeutic targets in parasitic nematodes (22) and in Trypanomastid parasites such as T. cruzi (11). This minireview will focus on new structural results derived from the recent crystal structure determinations of native E. coli PCK (23, 24), its complex with ATP-Mg-oxalate (25), and the implications for the active site residues and catalytic mechanism of E. coli and other ATPand GTP-dependent PCKs. We also suggest revised nucleotide-binding sites and possible active site residues for the GTP-dependent PCK family. Other aspects of GTP-dependent PCK enzymology and genetics have been recently reviewed (26).

Snapshot of an enzyme reaction intermediate in the structure of the ATP–Mg2+–oxalate ternary complex of Escherichia coli PEP carboxykinase

We report the 1.8 Å crystal structure of adenosine triphosphate (ATP)–magnesium–oxalate bound phosphoenolpyruvate carboxykinase (PCK) from Escherichia coli. ATP binding induces a 20° hinge-like rotation of the N- and C-terminal domains which closes the active-site cleft. PCK possesses a novel nucleotide-binding fold, particularly in the adenine-binding region, where the formation of a cis backbone torsion angle in a loop glycine residue promotes intimate contacts between the adenine-binding loop and adenine, while stabilizing a syn conformation of the base. This complex represents a reaction intermediate analogue along the pathway of the conversion of oxaloacetate to phosphoenolpyruvate, and provides insight into the mechanistic details of the chemical reaction catalysed by this enzyme.

Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents

Calcium-activated phosphoenolpyruvate carboxykinase fromEscheria coli is not inactivated by a number of sulfhydryl-directed reagents [5,5′-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N′-(5-sulfo-l-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5′-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.

Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phosphoenolpyruvate carboxykinases

Two members of the ATP-dependent class of phospho enol pyruvate (PEP) carboxykinases (Saccharomyces cerevisiae and Escherichia coli PEP carboxykinase), and one member of the GTP-dependent class (the cytosolic rat liver enzyme) have been comparatively analyzed by taking advantage of their intrinsic fluorescence. The S. cerevisiae and the rat liver enzymes show intrinsic fluorescence with a maximum emission characteristic of moderately buried tryptophan residues, while the E. coli carboxykinase shows somewhat more average exposure for these fluorophores. The fluorescence of the three proteins was similarly quenched by the polar compound acrylamide, but differences were observed for the ionic quencher iodide. For the ATP-dependent enzymes, these last experiments indicate more exposure to the aqueous media of the tryptophan population of the E. coli than of the S. cerevisiae enzyme. The effect of nucleotides on the emission intensities and quenching efficiencies revealed substrate-induced conformational changes in the E. coli and cytosolic rat liver PEP carboxykinases. The addition of Mn2+ or of the adenosine nucleotides in the presence of Mg2+ induced an enhancement in the fluorescence of the E. coli enzyme. The addition of guanosine or inosine nucleotides to the rat liver enzyme quenched its fluorescence. From the ligand-induced fluorescence changes, dissociation constants of 40 +/- 6 microM, 10 +/- 0.8 microM, and 15 +/- 1 microM were obtained for Mn2+, MgATP and MgADP binding to the E. coli enzyme, respectively. For the cytosolic rat liver PEP carboxykinase, the respective values for GDP, IDP and ITP binding are 6 +/- 0.5 microM, 6.7 +/- 0.4 microM and 10.1 +/- 1.7 microM. A comparison of the dissociation constants obtained in this work with those reported for other PEP carboxykinases is presented.

Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

Physical and genetic analysis of the phosphoenolpyruvate carboxykinase (pckA) locus from Escherichia coli K12

SummaryAn 8 kb BamHI fragment of the Escherichia coli K12 chromosome has been cloned which complemented the pheotype of CRM+pckA mutants with inactive phosphoenolpyruvate (PEP) carboxykinase. The pckA+ clones expressed levels of enzyme activity elevated up to 30-fold and produced a Mr 55000 product in maxicells, which coelectrophoresed with purified PEP carboxykinase. The cloned fragment expressed the pckA, ompR and envZ gene products in maxicells. The order of genes on the chromosome inferred from restriction mapping, was (74 min)...pckA, envZ ompR...(75 min). Transcription of the pckA, gene cloned on multicopy plasmids increased in stationary phase and was also regulated by catabolite repression. The transcriptional control region has been located by genetic fusions to the chloramphenicol acetyltransferase (cat) gene and pckA, was transcribed in the direction of envZ (clockwise direction on the chromosome).

Woodward's reagent K reacts with histidine and cysteine residues in Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases

The reaction of Woordwards reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Llamaset al. [(1986),J. Am. Chem. Soc.108, 5543–5548], but not with Sinha and Brewer [(1985),Anal. Biochem.151, 327–333]. The chemical modification ofEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein-WRK adducts.

Identification of reactive lysines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae

Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5′‐phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn2+. After digestion of the modified protein with trypsin plus protease V‐8, the labeled peptides were isolated by reverse‐phase high‐performance liquid chromatography and sequenced by gas‐phase automatic Edman degradation. Lys286 of bacterial PEPCK and Lys289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP‐dependent phosphoenolpyruvate carboxykinases described so far.

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12.

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.

Identification of reactive conserved histidines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae.

Escherichia coli and Saccharomyces cerevisiae phospho enol pyruvate (PEP) carboxykinases are inactivated by diethylpyrocarbonate (DEP). Inactivation follows pseudo-first-order kinetics and exhibits a second order rate constant of 0.8 M-1 s-1 for the bacterial enzyme and of 3.3 M-1 s-1 for the yeast carboxykinase. A mixture of ADP + PEP + MnCl2 protects against inactivation by DEP, suggesting that residues within the active site are being modified. After digestion of the modified proteins with trypsin, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by Edman degradation. His-271 of E. coli carboxykinase and His-273 of the yeast enzyme were identified as the reactive amino-acid residues. The modified histidine residues occupy equivalent positions in these enzymes, and they are located in a highly conserved region of all ATP-dependent phospho enol pyruvate carboxykinases described so far.