Louis T. J. Delbaere

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).

How do kinases transfer phosphoryl groups

Understanding how phosphoryl transfer is accomplished by kinases, a ubiquitous group of enzymes, is central to many biochemical processes. Qualitative analysis of the crystal structures of enzyme-substrate complexes of kinases reveals structural features of these enzymes important to phosphoryl transfer. Recently determined crystal structures which mimic the transition state complex have added new insight into the debate as to whether kinases use associative or dissociative mechanisms of catalysis.

The nerve of ovulation-inducing factor in semen

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12–23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC12) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo–pituitary–gonadal axis of the inseminated female.

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.

The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates.

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.

Comparison of the predicted model of |[alpha]|-lytic protease with the X-ray structure

THERE are several methods for the prediction of secondary structural features of protein molecules from the amino acid sequence1–5. Application of these methods to proteins of unknown tertiary structure has met with mixed results6,7, and it has been suggested that it is the protein structural type which will determine which predictive method will succeed8. There is also great interest in the ab initio prediction of tertiary structures of proteins9–11, but this has not yet been achieved. One possible method for predicting the tertiary structure of an enzyme is by the use of a known amino acid sequence and a previously determined tertiary structure of another related homologous isofunctional enzyme. This method was used by McLachlan and Shotton12 in an attempt to fit the α-lytic protease sequence into the polypeptide chain folding of elastase13 and chymotrypsin14. It has also been used to predict the tertiary structure of troponin C (ref. 15). Now that the structure of α-lytic protease is known16 to a resolution of 2.8 Å, we can assess the accuracy of such predictions. The tertiary structures of two related bacterial serine proteases, SGPA and SGPB and their structural relationship to the pancreatic enzymes have been published previously17–19. Many of the conclusions drawn from those studies are also applicable to the present discussion on α-lytic protease and need not be repeated. Rather, we shall consider the basic premise of sequence homology in phylogenetically distant proteins being used to deduce tertiary structures. A recent realignment20 of the amino acid sequences of Gly-Asp-Ser-Gly-Gly proteases, which was based on the known topological equivalences of α-carbon atoms, indicates an overall sequence identity of only 18% between α-lytic protease and elastase. We show here that this low value is independent of the environment of the topologically equivalent polypeptide chains (whether the residues are internal or external) and that the sequence identity is associated with only those residues which are in the immediate vicinity of the active site quartet, Asp 102, His 57, Ser 195 and Ser 214.

The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

BackgroundThe objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.MethodsSemen collected from male llamas by artificial vagina was centrifuged and the seminal plasma was harvested and stored frozen. Seminal plasma was thawed and loaded onto a Type 1 macro-prep ceramic hydroxylapatite column and elution was carried out using a lineal gradient with 350 mM sodium phosphate. Three protein fractions were identified clearly (Fractions A, B, and C), where a prominent protein band with a mass of 14 kDa was identified in Fraction C. Fraction C was loaded into a sephacryl gel filtration column for further purification using fast protein liquid chromatography (FPLC). Isocratic elution resulted in 2 distinct protein fractions (Fractions C1 and C2). An in vivo bioassay (n = 10 to 11 llamas per group) was used to determine the ovarian effect of each fraction involving treatment with saline (negative control), whole seminal plasma (positive control), or seminal plasma Fractions A, B or C2. Ultrasonography was done to detect ovulation and CL formation, and blood samples were taken to measure plasma progesterone and LH concentrations.ResultsOvulation and CL formation was detected in 0/10, 10/11, 0/10, 2/11, and 10/11 llamas treated with saline, whole seminal plasma, Fractions A, B and C2 respectively (P < 0.001). A surge in circulating concentrations of LH was detected within 2 hours only in llamas treated with either whole seminal plasma or Fraction C2. Plasma progesterone concentration and CL diameter profiles were greatest (P < 0.05) in llamas treated with Fraction C2.ConclusionOvulation-inducing factor was isolated from llama seminal plasma as a 14 kDa protein molecule that elicits a preovulatory LH surge followed by ovulation and CL formation in llamas, suggesting an endocrine effect at the level of the hypothalamus (release of GnRH) or the pituitary (gonadotrophs).

Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing.

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA29 ↓, releasing a Leu30 isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, 40QQTQSSKQQTPKIQ53. The preferred site is Gln43 with secondary processing at Gln47 and Gln53. This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu58 and then Val69 to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val69SspA promote rapid intermolecular processing of remaining zSspA at Glu65, producing an N-terminal 66HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His66 of this penultimate isoform blocks the active site by hydrogen bonding to Ser237 and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr232 proximal to Ser237, permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.

The 1·6 Å structure of histidine-containing phosphotransfer protein HPr from Streptococcus faecalis

The histidine-containing phosphocarrier protein (HPr) is a central component of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. The three-dimensional structure of Gram-positive Streptococcus faecalis HPr has been determined using the method of multiple isomorphous replacement. The R factor for all data is 0.156 for S. faecalis HPr at 1.6 A resolution with very good geometry. The overall folding topology of HPr is a classical open-faced beta-sandwich, consisting of four antiparallel beta-strands and three alpha-helices. Remarkable disallowed Ramachandran torsion angles of Ala16 at the active center, revealed by the X-ray structure of S. faecalis HPr, demonstrate a unique example of torsion-angle strain that is likely involved directly in protein function. A brief report concerning the torsion-angle strain has been presented recently. A newly-determined pH 7.0 structure is shown to have the same open conformation of the active center and the same torsion-angle strain at Ala16, suggesting that pH is not responsible for the structural observations. The current structure suggests a role for residues 12 and 51 in HPrs function, since they are involved in the active center through direct and indirect hydrogen-bonding interactions with the imidazole ring of His15. It is found that Ser46, the regulatory site in HPr from Gram-positive bacteria, N-caps the minor alpha-B helix and is also involved in the Asn43-Ser46 beta-turn. This finding, in conjunction with the proposed routes of communication between the regulatory site Ser46 and the active center in S. faecalis HPr, provides new insight into the understanding of how Ser46 might function. The putative involvement of the C-terminal alpha-carboxyl group and the related Gly67-Glu70 reverse beta-turn with respect to the function of HPr are described.