Substrate spectrum of L-rhamnulose kinase related to models derived from two ternary complex structures

The enzyme L-rhamnulose kinase from Escherichia coli participates in the degradation pathway of L-rhamnose, a common natural deoxy-hexose. The structure of the enzyme in a ternary complex with its substrates ADP and L-rhamnulose has been determined at 1.55A resolution and refined to R(cryst)/R(free) values of 0.179/0.209. The result was compared with the lower resolution structure of a corresponding complex containing L-fructose instead of L-rhamnulose. In light of the two established sugar positions and conformations, a number of rare sugars have been modeled into the active center of L-rhamnulose kinase and the model structures have been compared with the known enzymatic phosphorylation rates. Rare sugars are of rising interest for the synthesis of bioactive compounds.     

Conformational changes in the reaction of pyridoxal kinase

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat shock cognate 70/actin superfamily

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.     

Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP.phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.     

Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ～20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.     

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.     

Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6'' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.     

Binding and function of mitochondrial glycerol kinase in comparison with those of mitochondrial hexokinase

Mitochondrial-bound glycerol kinase in rat brain was examined with reference to factors involved in the binding and significance of the binding in relation to ATP metabolism inside the mitochondria. The mitochondrial-bound glycerol kinase was solubilized with glycerol 3-phosphate or ADP, and the solubilized enzyme was rebound to mitochondria by addition of divalent cations. The rebinding was decreased by the presence of glycerol 3-phosphate, while was increased by glucose 6-phosphate. Positive correlation was found between the formation of glycerol 3-phosphate by mitochondrial-bound glycerol kinase and ATP content in mitochondria in experiments using various concentrations of succinate and ADP. On the other hand, glycerol 3-phosphate formation was inhibited by addition of inhibitors for mitochondria functions, such as oligomycin, dinitrophenol, cyanide, and atractyloside. Furthermore, formation of dihydroxyacetone phosphate from glycerol was proved, indicating the involvement of glycerol kinase in glycerol phosphate shuttle in combination with glycerol phosphate dehydrogenase. These findings are discussed in comparison with those of mitochondrial-bound hexokinase.     

Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Leu(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K(m) values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.     

The 2.1 A structure of Torpedo californica creatine kinase complexed with the ADP-Mg(2+)-NO(3)(-)-creatine transition-state analogue complex

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.     

Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures

5'-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96 degrees domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor alpha,beta-methylene ADP, have been determined at 2.1 A and 1.85 A resolution, respectively. In addition, a complex of the open form of 5'-nucleotidase with ATP was analyzed at a resolution of 1.7 A. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The beta-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.     

Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase

Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.     

Crystal structure and substrate specificity of plant adenylate isopentenyltransferase from Humulus lupulus: distinctive binding affinity for purine and pyrimidine nucleotides

Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA–IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA–IPTs act on specific sites of tRNA. Here, we present the crystal structure of an AIPT–ATP complex from Humulus lupulus (HlAIPT), which is similar to the previous structures of Agrobacterium AIPT and yeast tRNA–IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ∼ ADP > GTP > CTP > UTP. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA–IPT, HlAIPT has evolved with a different binding strategy for adenylate.     

Structural insights into substrate specificity of crotonase from the n-butanol producing bacterium Clostridium acetobutylicum

Crotonase from Clostridium acetobutylicum (CaCRT) is an enzyme that catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway. To investigate the molecular mechanism underlying n-butanol biosynthesis, we determined the crystal structures of the CaCRT protein in apo- and acetoacetyl-CoA bound forms. Similar to other canonical crotonase enzymes, CaCRT forms a hexamer by the dimerization of two trimers. A crystal structure of CaCRT in complex with acetoacetyl-CoA revealed that Ser69 and Ala24 to be signature residues of CaCRT, which results in a distinct ADP binding mode wherein the ADP moiety is bound at a different position compared with other crotonases. We also revealed that the substrate specificity of crotonase enzymes is determined by both the structural feature of the α3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed α3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. The key residues involved in substrate binding, enzyme catalysis and substrate specificity were confirmed by site-directed mutagenesis.     

Ligand-induced structural changes in adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step, ATP-dependent conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS serves as the sulfuryl donor for the biosynthesis of all sulfate esters and also as a precursor of reduced sulfur biomolecules in many organisms. Previously, we determined the crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum [MacRae et al. (2000) Biochemistry 39, 1613-1621]. That structure contained a protease-susceptible disordered region ("mobile lid"; residues 145-170). Addition of MgADP and APS, which together promote the formation of a nonproductive "dead-end" ternary complex, protected the lid from trypsin. This report presents the 1.43 A resolution crystal structure of APS kinase with both ADP and APS bound at the active site and the 2.0 A resolution structure of the enzyme with ADP alone bound. The mobile lid is ordered in both complexes and is shown to provide part of the binding site for APS. That site is formed primarily by the highly conserved Arg 66, Arg 80, and Phe 75 from the protein core and Phe 165 from the mobile lid. The two Phe residues straddle the adenine ring of bound APS. Arg 148, a completely conserved residue, is the only residue in the mobile lid that interacts directly with bound ADP. Ser 34, located in the apex of the P-loop, hydrogen-bonds to the 3'-OH of APS, the phosphoryl transfer target. The structure of the binary E.ADP complex revealed further changes in the active site and N-terminal helix that occur upon the binding/release of (P)APS.     

Study of pH-dependence of kinetic parameters of pyruvate kinase from bovine adrenal cortex

The effect of pH on the main kinetic parameters of pyruvate kinase function was studied. The maximal rate of the reaction as well as the values of Km for ADP and Ki for phenylalanine depend on pH and show a well-defined extremum at pH 6.8-7.0. Spectrofluorimetric titration of pyruvate kinase results in pH dependencies of changes in the fluorescence spectra parameters (e.g., quantum yield, half-width and position of the maximum). This enabled to determine the pH regions corresponding to changes in the state of tryptophan residues. Data from the enzyme inhibition by phenylalanine suggest that acidification of the medium leads to the decrease of the catalytic activity due to the protonation of the ionogenic group of the enzyme. Within the pH range of 7.0-8.0, the decrease of the pyruvate kinase activity is due to structural shifts in the enzyme molecule, as a result of which the steric complementariness of the enzyme active center with respect to the substrate (Mg.ADP) is impaired.     

Phosphofructokinase: structure and control

Phosphofructokinase from Bacillus stearothermophilus shows cooperative kinetics with respect to the substrate fructose-6-phosphate (F6P), allosteric activation by ADP, and inhibition by phosphoenolpyruvate. The crystal structure of the active conformation of the enzyme has been solved to 2.4 A resolution, and three ligand-binding sites have been located. Two of these form the active site and bind the substrates F6P and ATP. The third site binds both allosteric activator and inhibitor. The complex of the enzyme with F6P and ADP has been partly refined at 2.4 A resolution, and a model of ATP has been built into the active site by using the refined model of ADP and a 6 A resolution map of bound 5'-adenylylimidodiphosphate (AMPPNP). The gamma-phosphate of ATP is close to the 1-hydroxyl of F6P, in a suitable position for in-line phosphoryl transfer. The binding of the phosphate of F6P involves two arginines from a neighbouring subunit in the tetramer, which suggests that a rearrangement of the subunits could explain the cooperativity of substrate binding. The activatory ADP is also bound by residues from two subunits.     

The roles of ADP2- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme. A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site. From the change in chemical shifts of the 'basic patch' histidine resonances (His62, 167 and 170), the nucleotide C8-H, C2-H and C1'-H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e. catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site. A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups. The presence of Mg2+ results in further differences being observed. The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the 'basic patch' region of the N-terminal domain. In the presence of greater than or equal to 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site. Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP). It is suggested that alpha-helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg . ATP, consistent with Mg2+ assisting the transfer of the gamma-phosphate of ATP to 3-phosphoglycerate during catalysis.     

Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase

Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.