Ordered Binding Mechanism Research Articles

New 4-Amino-1,2,3-Triazole Inhibitors of Indoleamine 2,3-Dioxygenase Form a Long-Lived Complex with the Enzyme and Display Exquisite Cellular Potency.

Indoleamine-2,3 dioxygenase 1 (IDO1) has emerged as a central regulator of immune responses in both normal and disease biology. Due to its established role in promoting tumour immune escape, IDO1 has become an attractive target for cancer treatment. A novel series of highly cell potent IDO1 inhibitors based on a 4-amino-1,2,3-triazole core have been identified. Comprehensive kinetic, biochemical and structural studies demonstrate that compounds from this series have a noncompetitive kinetic mechanism of action with respect to the tryptophan substrate. In co-complex crystal structures, the compounds bind in the tryptophan pocket and make a direct ligand interaction with the haem iron of the porphyrin cofactor. It is proposed that these data can be rationalised by an ordered-binding mechanism, in which the inhibitor binds an apo form of the enzyme that is not competent to bind tryptophan. These inhibitors also form a very tight, long-lived complex with the enzyme, which partially explains their exquisite cellular potency. This novel series represents an attractive starting point for the future development of potent IDO1-targeted drugs.

Determination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae.

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 μM) bound before isopentenyl diphosphate (KM =40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus.

Functional Characterization of SdcF from Bacillus licheniformis, a Homolog of the SLC13 Na+/Dicarboxylate Transporters

The SdcF transporter from Bacillus licheniformis (gene BL02343) is a member of the divalent anion sodium symporter (DASS)/SLC13 family that includes Na⁺/dicarboxylate transporters from bacteria to humans. SdcF was functionally expressed in Escherichia coli (BL21) and assayed in right side out membrane vesicles. ScdF catalyzed the sodium-coupled transport of succinate and α-ketoglutarate. Succinate transport was strongly inhibited by malate, fumarate, tartrate, oxaloacetate and L-aspartate. Similar to the other DASS transporters, succinate transport by SdcF was inhibited by anthranilic acids, N-(p-amylcinnamoyl) anthranilic acid and flufenamate. SdcF transport was cation-dependent, with a K₀.₅ for sodium of ~1.5 mM and a K₀.₅ for Li⁺ of ~40 mM. Succinate transport kinetics by SdcF were sigmoidal, suggesting that SdcF may contain two cooperative substrate binding sites. The results support an ordered binding mechanism for SdcF in which sodium binds first and succinate binds last. We conclude that SdcF is a secondary active transporter for four- and five-carbon dicarboxylates that can use Na⁺ or Li⁺ as a driving cation.

Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

A COMPARISON BETWEEN THE INITIAL RATE EXPRESSIONS OBTAINED UNDER STRICT CONDITIONS AND THE RAPID EQUILIBRIUM ASSUMPTION USING, AS EXAMPLE, A FOUR SUBSTRATE ENZYME REACTION

The software WinStes, developed by our group, is used to derive the strict steady-state initial rate equation of the reaction mechanism of CTP:sn-glycerol-3-phosphate cytidylyltransferase [EC 2.7.7.39] from Bacillus subtilis. This enzyme catalyzes a reaction with two substrates and operates by a random ordered binding mechanism with two molecules of each substrate. The accuracy of the steady-state rate equation derived is checked by comparing the rate values it provides with those obtained from the simulated progress curves. To analyze the kinetics of this enzyme using the strict steady-state initial rate equation, several curves for different substrate concentrations and different rate constants are generated. A comparison of these curves with the curves obtained from the rapid equilibrium initial rate equation, with different substrate concentration values, serves to analyze how the strict steady-state rate equation values are closer to those of rapid equilibrium rate equations when rapid equilibrium conditions are fulfilled.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr286-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr286 autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

Obligate Ordered Binding of Human Lactogenic Cytokines

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.

Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis

Trytophan Hydroxylase Type I (TPH1), most abundantly expressed in the gastrointestinal tract, initiates the synthesis of serotonin by catalyzing hydroxylation of tryptophan in the presence of biopterin and oxygen. We have previously described three series of novel, periphery-specific TPH1 inhibitors that selectively deplete serotonin in the gastrointestinal tract. We have now determined co-crystal structures of TPH1 with three of these inhibitors at high resolution. Analysis of the structural data showed that each of the three inhibitors fills the tryptophan binding pocket of TPH1 without reaching into the binding site of the cofactor pterin, and induces major conformational changes of the enzyme. The enzyme-inhibitor complexes assume a compact conformation that is similar to the one in tryptophan complex. Kinetic analysis showed that all three inhibitors are competitive versus the substrate tryptophan, consistent with the structural data that the compounds occupy the tryptophan binding site. On the other hand, all three inhibitors appear to be uncompetitive versus the cofactor 6-methyltetrahydropterin, which is not only consistent with the structural data but also indicate that the hydroxylation reaction follows an ordered binding mechanism in which a productive complex is formed only if tryptophan binds only after pterin, similar to the kinetic mechanisms of tyrosine and phenylalanine hydroxylase.

Conformational changes associated with cofactor/substrate binding of 6-phosphogluconate dehydrogenase from Escherichia coli and Klebsiella pneumoniae: Implications for enzyme mechanism

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP(+) to NADPH and the release of CO(2). Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10 degrees rotation and resulting in a 7A movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP(+)-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues.

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases.

Carbamoyl phosphate (CP) has a half-life for thermal decomposition of <2 s at 100 degrees C, yet this critical metabolic intermediate is found even in organisms that grow at 95-100 degrees C. We show here that the binding of CP to the enzymes aspartate and ornithine transcarbamoylase reduces the rate of thermal decomposition of CP by a factor of >5,000. Both of these transcarbamoylases use an ordered-binding mechanism in which CP binds first, allowing the formation of an enzyme.CP complex. To understand how the enzyme.CP complex is able to stabilize CP we investigated the mechanism of the thermal decomposition of CP in aqueous solution in the absence and presence of enzyme. By quantum mechanics/molecular mechanics calculations we show that the critical step in the thermal decomposition of CP in aqueous solution, in the absence of enzyme, involves the breaking of the C O bond facilitated by intramolecular proton transfer from the amine to the phosphate. Furthermore, we demonstrate that the binding of CP to the active sites of these enzymes significantly inhibits this process by restricting the accessible conformations of the bound ligand to those disfavoring the reactive geometry. These results not only provide insight into the reaction pathways for the thermal decomposition of free CP in an aqueous solution but also show why these reaction pathways are not accessible when the metabolite is bound to the active sites of these transcarbamoylases.

ATP-conjugated peptide inhibitors for calmodulin-dependent protein kinase II

Substrate analog peptides of CaMKII with varying degrees of the inhibitory potency were linked to ATPγS either by considering a phosphoryl transfer mechanism or simply by using a relatively long flexible linker. The latter bisubstrate inhibitors showed relatively little effects while the former ones improved inhibitory potency to different levels depending on the binding affinities of the peptide moieties. One of the mechanism-based bisubstrate inhibitors was then utilized to demonstrate an ATP-competitive but peptide substrate-uncompetitive inhibition, supporting an ordered binding mechanism for CaMKII.

Phosphoryl Transfer Is Not Rate-Limiting for the ROCK I-Catalyzed Kinase Reaction

Rho-associated coiled-coil kinase, ROCK, is implicated in Rho-mediated cell adhesion and smooth muscle contraction. Animal models suggest that the inhibition of ROCK can ameliorate conditions, such as vasospasm, hypertension, and inflammation. As part of our effort to design novel inhibitors of ROCK, we investigated the kinetic mechanism of ROCK I. Steady-state bisubstrate kinetics, inhibition kinetics, isotope partition analysis, viscosity effects, and presteady-state kinetics were used to explore the kinetic mechanism. Plots of reciprocals of initial rates obtained in the presence of nonhydrolyzable ATP analogues and the small molecule inhibitor of ROCK, Y-27632, against the reciprocals of the peptide concentrations yielded parallel lines (uncompetitive pattern). This pattern is indicative of an ordered binding mechanism, with the peptide adding first. The staurosporine analogue K252a, however, gave a noncompetitive pattern. When a pulse of (33)P-gamma-ATP mixed with ROCK was chased with excess unlabeled ATP and peptide, 0.66 enzyme equivalent of (33)P-phosphate was incorporated into the product in the first turnover. The presence of ATPase activity coupled with the isotope partition data is a clear evidence for the existence of a viable [E-ATP] complex in the kinase reaction and implicates a random binding mechanism. The k(cat)/K(m) parameters were fully sensitive to viscosity (viscosity effects of 1.4 +/- 0.2 and 0.9 +/- 0.3 for ATP and peptide 5, respectively), and therefore, the barriers to dissociation of either substrate are higher than the barrier for the phosphoryl transfer step. As a consequence, not all the binding steps are at fast equilibrium. The observation of a burst in presteady-state kinetics (k(b) = 10.2 +/- 2.1 s(-)(1)) and the viscosity effect on k(cat) of 1.3 +/- 0.2 characterize the phosphoryl transfer step to be fast and the release of product and/or the enzyme isomerization step accompanying it as rate-limiting at V(max) conditions. From the multiple kinetic studies, most of the rate constants for the individual steps were either evaluated or estimated.

Charge Translocation During Cosubstrate Binding in the Na +/Proline Transporter of E. coli

Charge translocation associated with the activity of the Na(+)/proline cotransporter PutP of Escherichia coli was analyzed for the first time. Using a rapid solution exchange technique combined with a solid-supported membrane (SSM), it was demonstrated that Na(+)and/or proline individually or together induce a displacement of charge. This was assigned to an electrogenic Na(+)and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k>50s(-1) which preceeds the rate-limiting step. Based on the kinetic analysis of our electrical signals, the following characteristics are proposed for substrate binding in PutP. (1) Substrate binding is electrogenic not only for Na(+), but also for the uncharged cosubstrate proline. The charge displacement associated with the binding of both substrates is of comparable size and independent of the presence of the respective cosubstrate. (2) Both substrates can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails, while at sufficiently high concentrations, each substrate can bind in the absence of the other. (3) Both substrate binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate. (4) Proline binding proceeds in a two-step process: low affinity (approximately 1mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition.

Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB.

Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S]2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S]2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S]2+ cluster and the [4Fe-4S]2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at approximately 400 nm that we attribute to changes in the coordination environment of the [4Fe-4S]2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5'-deoxyadenosyl radicals in the absence of the correct substrate.

The sodium/substrate symporter family: structural and functional features

Members of the sodium/substrate symporter family (SSSF, TC 2.A.21) catalyze the uptake of a wide variety of solutes including sugars, proline, pantothenate, and iodide into cells of pro- and eukaryotic origin. Extensive analyses of the topology of different SSSF proteins suggest an arrangement of 13 transmembrane domains as a common topological motif. Regions involved in sodium and/or substrate binding were identified. Furthermore, protein chemical and spectroscopic studies reveal ligand-induced structural alterations which are consistent with close interactions between the sites of sodium and substrate binding, thereby supporting an ordered binding mechanism for transport.

Activation of Na +,K +,Cl − cotransport in squid giant axon by extracellular ions: evidence for ordered binding

Activation of the influx mode of the Na +,K +,Cl − cotransporter (NKCC) by extracellular Na +, K + and Cl − was studied using the internally dialyzed squid giant axon. Cooperative interactions among the three transported ions were assessed using ion activation of NKCC-mediated 36Cl influx under two sets of experimental conditions. The first, or control condition, used high, non-limiting concentrations of two of the cotransported ions (the co-ions) while activating cotransport with the third ion. Under this non-limiting co-ion condition the calculated V max of the cotransporter was between 57 and 60 pmol/cm 2/s. The apparent activation ( K App, or half-saturation) constants were: K +, 9 mM; Na +, 52 mM; and Cl −, 146 mM. The second condition used limiting co-ion concentration conditions. In this case, activation by each ion was determined when one of the other two co-ions was present at or near its apparent half-saturation concentration as determined above. Under these limiting conditions, the K App values for all three co-ions were significantly increased regardless of which co-ion was present at a limiting concentration. The effects on the apparent V max were more complicated. When K + was the limiting co-ion, there was little effect on the V max for Na + or Cl − activation. In contrast, limiting concentrations of Na + or Cl − both resulted in a large reduction of the apparent V max when activating with the other two co-ions. These results are consistent with an ordered binding mechanism for the NKCC in which K + binds before Na + or Cl −. Physiological implications for these results are discussed.

Antibody-Catalyzed Decarboxylative Oxidation of Vanillylmandelic Acid

The most important industrial process for the synthesis of vanillin is performed in two steps involving an electrophilic aromatic substitution of glyoxylic acid on guaiacol followed by an oxidative decarboxylation of the intermediary α-hydroxy acids formed, thereby producing not only vanillin, but also byproducts which have to be eliminated. In the present study, we took advantage of the high specificity of catalytic antibodies to improve the synthesis of vanillin. Among 11 monoclonal antibodies elicited against the quaternary ammonium hapten H3, antibody H3-12 was found to catalyze the oxidative decarboxylation of vanillylmandelic acid (VMA), the precursor of vanillin, in the presence of sodium metaperiodate. The kinetic data of the antibody-catalyzed reaction are consistent with an ordered binding mechanism. At pH 9.0, H3-12 catalyzed the transformation of VMA into vanillin with a kcat of 2.70 min-1, a Michaelis−Menten constant Ka for the binary complex of 260 μM, and a Kb for the ternary complex of 2100 μM. The catalyzed reaction was fully inhibited by a hapten analogue with a Ki of 10 μM. The fine specificity of anti-H3 monoclonal antibodies was determined using H3-related compounds with a competitive enzyme immunoassay. Controls demonstrating that catalytic activity is actually related to antibody binding, and mechanistic studies, are also presented.

Kinetic Analysis of the Catalytic Mechanism of Serotonin N-Acetyltransferase (EC 2.3.1.87)

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin biosynthesis. This enzyme is of special biological interest because large changes in its activity drive the large night/day rhythm in circulating melatonin in vertebrates. In this study the kinetic mechanism of AANAT action was studied using bacterially expressed glutathione S-transferase (GST)-AANAT fusion protein. The enzymologic behavior of GST-AANAT and cleaved AANAT was essentially identical. Two-substrate kinetic analysis generated an intersecting line pattern characteristic of a ternary complex mechanism. The dead end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus tryptamine. Tryptophol was not an alternative substrate but was a dead end competitive inhibitor versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acetyl-CoA first. N-Acetyltryptamine, a reaction product, was a noncompetitive inhibitor versus tryptamine and uncompetitive with respect to acetyl-CoA. Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framework for inhibitor design.

Yeast protein geranylgeranyltransferase type-I: steady-state kinetics and substrate binding.

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in Leu or Phe by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). A kinetic study of the alkylation reaction was conducted with a continuous assay based on the fluorescence enhancement that accompanies geranylgeranylation of dansyl-GCIIL. The kinetic constants k(cat) = 0.34 +/- 0.01 s(-1), K(M)(G) = 0.86 +/- 0.05 microM for GGPP, and K(M)(D) = 1.6 +/- 0.1 microM for dansyl-GCIIL were calculated from initial rates measured at varying concentrations of the substrates. Inhibitor studies were conducted with dead-end inhibitors for GGPP and the peptide substrate. Double reciprocal plots for the peptide mimic Cys-AMBA-Leu gave a competitive pattern when plotted against varying concentrations of dansyl-GCIIL and an uncompetitive pattern against GGPP. Similar plots for 1-phosphono-(E,E,E)-geranylgeraniol, a dead-end inhibitor for GGPP, gave a competitive double reciprocal plot for varied concentrations of GGPP and induced potent substrate inhibition by dansyl-GCIIL when dansyl-GCIIL was the varied substrate. The dissociation constant (K(D)) for the PGGTase-I x GGPP complex was 120 +/- 20 nM. These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.

The Sequencing, Expression, Purification, and Steady-State Kinetic Analysis of Quinolinate Phosphoribosyl Transferase fromEscherichia coli

ThenadCgene fromEscherichia coliwas isolated and sequenced. The gene was then cloned into an expression vector and, following transformation, the resulting bacteria were able to produce quinolinate phosphoribosyl transferase as about 2% of the soluble protein. The enzyme was purified in five steps leading to a homogeneous preparation. The enzyme reaction shows an ordered binding mechanism where the magnesium ion complex of 5-phosphoribosyl-1-pyrophosphate binds first followed by quinolinic acid. The products are pyrophosphate, CO2, and nicotinate mononucleotide. Product inhibition studies show that nicotinate mononucleotide is a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate while pyrophosphate is noncompetitive with respect to both 5-phosphoribosyl-1-pyrophosphate and quinolinic acid. Phthalic acid and fructose-1,6-bisphosphate were used as dead-end inhibitors. Phthalate was competitive with respect to quinolinic acid but uncompetitive with respect to 5-phosphoribosyl-1-pyrophosphate. Fructose-1,6-bisphosphate was a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate and noncompetitive with respect to quinolinic acid. TheKmvalues for the substrates are 15.6 μMfor 5-phosphoribosyl-1-pyrophosphate and 6.4 μMfor quinolinic acid.