Functional evaluation of serine/threonine residues in the P-Loop of Rhodobacter sphaeroides phosphoribulokinase.

The N-terminal region of phosphoribulokinase (PRK) has been proposed to contain a "P-loop" or "Walker A" motif. In Rhodobacter sphaeroides PRK, four alcohol side chains, contributed by S14, T18, S19, and T20, map within the P loop and represent potential Mg-ATP ligands. Each of these has been individually replaced with an alanine and the impact of these substitutions on enzyme-ATP interactions and overall catalytic efficiency evaluated. Each mutant PRK retains the ability to tightly bind the positive effector, NADH (0.7-0.9 per site), and exhibits allosteric activation, suggesting that the proteins retain a high degree of structural integrity. Similarly, each mutant PRK retains the ability to stoichiometrically (0.7-1.2 per site) bind the alternative substrate trinitrophenyl-ATP. Despite the large size of the PRK oligomer (8 x 32 kDa), (31)P NMR can be used to detect stoichiometrically bound Mg-ATP substrate, which produces markedly broadened peaks in comparison with signals from unbound Mg-ATP. Elimination of alcohol substituents in mutants T18A, S19A, or T20A produces enzymes which retain the ability to form stable PRKMg-ATP complexes. Each mutant complex is characterized by (31)P resonances for alpha- and gamma-phosphoryls of bound Mg-ATP which are narrower than measured for wild-type PRKMg-ATP; signals for the beta-phosphoryl are poorly detectable for mutant PRKMg-ATP complexes. Kinetic characterization indicates that these mutants differ markedly with respect to catalytic activity. T20A exhibits V(m) comparable to wild-type PRK, while V(m) is diminished by 8-fold for T18A and by 40-fold for S14A. In contrast to these modest effects, S19A exhibits decreases in V(m) and V(m)/K(Ru5P) of 500-fold and >15000-fold, respectively. S19A and T18A exhibit only modest (6-7-fold) increases in S(1/2) for ATP but larger (30-45-fold) increases in K(m) for Ru5P. K(I) values for the competitive inhibitor, 6-phosphogluconate, do not significantly change upon mutation of T18 or S19, suggesting that these residues are not crucial to Ru5P binding. A role for the alcohol group of S19, the eighth residue in P-loop motif, as a ligand to the Mg-ATP substrate seems compatible with the characterization data; adjacent alcohols do not efficiently function as surrogates. Such a proposed function for S19 is compatible with its proximity to E131, the acidic residue in a putative Walker B motif and probable second Mg-ATP ligand in PRK's active site.

Characterization of mevalonate kinase V377I, a mutant implicated in defective isoprenoid biosynthesis and HIDS/periodic fever syndrome.

The list of diseases linked to defects in lipid metabolism has recently been augmented by the addition of hyperimmunoglobulinemia D and periodic fever syndrome (HIDS: MIM 260920), which are correlated with depressed levels of mevalonate kinase activity [1,2] and protein [1]. More specifically, a V377I substitution has been proposed to account for this disease. We observed that V377 appears to be far from invariant in eukaryotic mevalonate kinases. Prokaryotic mevalonate kinases are lower in molecular weight and several terminate prior to residue 377 of the eukaryotic proteins. These observations prompted our direct test of the impact of V377 on activity and protein stability by engineering a V377I mutation in a recombinant human mevalonate kinase. The mutant protein has been isolated and kinetically characterized. In comparison with wild-type enzyme, V377I exhibits only modest differences (notably > or = 6-fold inflation of K(m(MVA))) that do not account for the diminished mevalonate kinase activity assayed in HIDS cell extracts. Moreover, thermal inactivation (50 degrees C) of isolated wild-type and V377I enzymes demonstrates little difference in stability between these proteins. We conclude that a single V377I substitution is unlikely to explain the observation of depressed mevalonate kinase stability and catalytic activity in HIDS.

Investigation of invariant serine/threonine residues in mevalonate kinase. Tests of the functional significance of a proposed substrate binding motif and a site implicated in human inherited disease.

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in K(m)((ATP)). K(m) for mevalonate increases by approximately 20-fold for S146A, approximately 40-fold for T243A, and 100-fold for S201A. V(max) changes for S145A, S201A, and T243A are < or =3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V(max) for S146A is diminished by 4000-fold. In terms of V/K(MVA), this substitution produces a 10(5)-fold effect, suggesting an active site location and catalytic role for Ser-146. The large k(cat) effect suggests that Ser-146 productively orients ATP during catalysis. K(D(Mg-ATP)) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg(2+)-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.

3-Hydroxy-3-methylglutaryl-CoA synthase: participation of invariant acidic residues in formation of the acetyl-S-enzyme reaction intermediate.

Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a concentration-dependent and substrate-protectable manner suggested that the active site contains reactive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characterization of the isolated synthase variants' ability to catalyze overall and partial reactions identified three mutant synthases (D99A, D159A, and D203A) that exhibit significant diminution of k(cat) for the overall reaction (10(2)-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7. 0 and 22 degrees C) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rate-limiting status. Differences in substrate saturation do not account for impaired activities or rates of intermediate formation. The structural integrity of the purified mutants' active sites is demonstrated by their abilities to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and stoichiometries comparable to values measured for wild-type synthase. The impact of three distinct amino acids on reaction intermediate formation supports a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. (18)O-induced shift of the (13)C NMR signal of (13)C acetyl-S-enzyme demonstrates that an analogous tetrahedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit retarded kinetics of solvent (18)O exchange while D99A fails to support (18)O exchange.

Phosphoribulokinase: current perspectives on the structure/function basis for regulation and catalysis.

Phosphoribulokinase (PRK), an enzyme unique to the reductive pentose phosphate pathway of CO2 assimilation, exhibits distinctive contrasting properties when the proteins from eukaryotic and prokaryotic sources are compared. The eukaryotic PRKs are typically dimers of -39 kDa subunits while the prokaryotic PRKs are octamers of -32 kDa subunits. The enzymes from these two classes are regulated by different mechanisms. Thioredoxin of mediated thiol-disulfide exchange interconverts eukaryotic PRKs between reduced (active) and oxidized (inactive) forms. Allosteric effectors, including activator NADH and inhibitors AMP and phosphoenolpyruvate, regulate activity of prokaryotic PRK. The effector binding site has been identified in the high resolution structure recently elucidated for prokaryotic PRK and the7 apparatus for transmission of the allosteric stimulus has been identified. Additional contrasts between PRKs include marked differences in primary structure between eukaryotic and prokaryotic PRKs. Alignment of all available deduced PRK sequences indicates that less than 10% of the amino acid residues are invariant. In contrast to these differences, the mechanism for ribulose 1,5-biphosphate synthesis from ATP and ribulose 5-phosphate (Ru5P) appears to be the same for all PRKs. Consensus sequences associated with M++-ATP binding, identified in all PRK proteins, are closely juxtaposed to the residue proposed to function as general base catalyst. Sequence homology and mutagenesis approaches have suggested several residues that may potentially function in Ru5P binding. Not all of these proposed Ru5P binding residues are closely juxtaposed in the structure of unliganded PRK. Mechanistic approaches have been employed to investigate the amino acids which influence K(m Ru5P) and identify those amino acids most directly involved in Ru5P binding. PRK is one member of a family of phospho or sulfo transferase proteins which exhibit a nucleotide monophosphate kinase fold. Structure/function correlations elucidated for PRK suggest analogous assignments for other members of this family of proteins.

3-Hydroxy-3-methylglutaryl-CoA synthase. A role for glutamate 95 in general acid/base catalysis of C-C bond formation.

Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by (13)C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu(95) functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK(a) of Glu(95) as required to support its function as a general acid.

3-hydroxy-3-methylglutaryl-coenzyme A synthase reaction intermediates: detection of a covalent tetrahedral adduct by differential isotope shift 13C nuclear magnetic resonance spectroscopy.

Binding of [1,2-(13)C]acetyl-CoA to wild-type 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is characterized by large upfield shifts for C1 (184 ppm, Deltadelta = 20 ppm) and C2 (26 ppm, Deltadelta = 7 ppm) resonances that are attributable to formation of the covalent [1,2 -(13)C]acetyl-S-enzyme reaction intermediate. NMR spectra of [1, 2-(13)C]acetyl-S-enzyme prepared in H(2)(16)O versus H(2)(18)O indicate a 0.055 ppm upfield shift of the C1 resonance in the presence of the heavier isotope. The magnitude of this (18)O-induced (13)C shift suggests that the 184 ppm resonance is attributable to a reaction intermediate in which C1 exhibits substantial carbonyl character. No significant shift of the C2 resonance occurs. These observations suggest that, in the absence of second substrate (acetoacetyl-CoA), enzymatic addition of H(2)(18)O to the C1 carbonyl of acetyl-S-enzyme occurs to transiently produce a tetrahedral species. This tetrahedral adduct exchanges oxygen upon backward collapse to re-form the sp(2)-hybridized thioester carbonyl. In contrast with HMG-CoA synthase, C378G Zoogloea ramigera beta-ketothiolase, which also forms a (13)C NMR-observable covalent acetyl-enzyme species, exhibits no (18)O-induced shift. Formation of the [(13)C]acetyl-S-enzyme reaction intermediate of HMG-CoA synthase in D(2)O versus H(2)O is characterized by a time-dependent isotope-induced upfield shift of the C1 resonance (maximal shift = 0. 185 ppm) in the presence of the heavier isotope. A more modest upfield shift (0.080 ppm) is observed for C378G Z. ramigera beta-ketothiolase in similar experiments. The slow kinetics for the development of the deuterium-induced (13)C shift in the HMG-CoA synthase experiments suggest a specific interaction (hydrogen bond) with a slowly exchangeable proton (deuteron) of a side chain/backbone of an amino acid residue at the active site.

Identification of the allosteric regulatory site in bacterial phosphoribulokinase.

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.

Rhodobacter sphaeroides phosphoribulokinase: identification of lysine-165 as a catalytic residue and evaluation of the contributions of invariant basic amino acids to ribulose 5-phosphate binding.

Rhodobacter sphaeroides phosphoribulokinase (PRK) is inactivated upon exposure to pyridoxal phosphate/sodium borohydride, suggesting a reactive lysine residue. Protection is afforded by a combination of the substrate ATP and the allosteric activator NADH, suggesting that the targeted lysine maps within the active site. PRK contains two invariant lysines, K53 and K165. PRK-K53M retains sensitivity to pyridoxal phosphate, implicating K165 as the target of this reagent. PRK-K165M retains wild-type structure, as judged by titration with effector NADH and the tight-binding alternative substrate trinitrophenyl-ATP. The catalytic activity of K165M and K165C mutants is depressed by >10(3)-fold. Residual activity of K165M is insensitive to pyridoxal phosphate, confirming K165 as the target of this reagent. The decreased catalytic efficiency of K165 mutants approaches the effect measured for a mutant of D169, which forms a salt-bridge to K165. K165M exhibits a 10-fold increase in S()1(/)()2 (ATP) and a 10(2)-fold increase in K(m) (Ru5P). To evaluate the contribution to Ru5P binding of K165 in comparison with this substrate's interaction with invariant H45, R49, R168, and R173, PRKs mutated at these positions have been used to determine relative K(i) values for 6-phosphogluconate, a competitive inhibitor with respect to Ru5P. Elimination of the basic side chain of K165, R49, and H45 results in increases in K(m) (Ru5P) which correlate well with the magnitude of increases in K(i) (phosphogluconate). In contrast, while mutations eliminating charge from R168 and R173 result in enzymes with substantial increases in K(m) (Ru5P), such mutant enzymes exhibit only small increases in K(i) (phosphogluconate). These observations suggest that K165, R49, and H45 are major contributors to Ru5P binding.

The crystal structure of phosphoribulokinase from Rhodobacter sphaeroides reveals a fold similar to that of adenylate kinase.

The essential photosynthetic enzyme phosphoribulokinase (PRK) is responsible for the conversion of ribulose 5-phosphate (Ru5P) to ribulose 1,5-bisphosphate, the substrate for the CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). We have determined the structure of the octameric bacterial form of PRK to a resolution of 2.5 A. The protein is folded into a seven-member mixed beta-sheet surrounded by alpha-helices, giving the overall appearance of the nucleotide monophosphate family of kinases. Homology with the nucleotide monophosphate kinases suggests a number of amino acid residues that are likely to be important in catalysis and suggests the roles of some amino acid residues that have been mutated prior to the determination of the structure. Further, sequence identity across eukaryotic and prokaryotic species and a calculation of the buried surface area suggests the identity within the octamer of a dimer conserved throughout evolution. The width of the groove leading to the active site is consistent with an oriented molecule of thioredoxin controlling the oxidation state of two cysteines that regulate activity in the eukaryotic enzymes. Although neither Asp 42 nor Asp 169 can be definitively assigned as the catalytic base, the crystal structure suggests the location of a ribulose 5-phosphate binding site and suggests a role for several of the conserved basic residues.

Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase.

Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, A., and Miziorko, H. M. (1998) Biochemistry (submitted for publication)] reveals that it folds in a manner similar to that of adenylate kinase. Three invariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previously functionally evaluated. These arginine residues map within the mobile lid domain that is a distinctive feature of the adenylate kinase family of proteins. Precedent for the significant function of arginines in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines. Solution state characterization of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiometric binding of an alternative nucleotide substrate (trinitrophenyl-ATP) as well as the allosteric effector (NADH). Kinetic characterization indicated > 10(4)-fold diminution in V/KRu5P for R168Q, attributable to a > 300-fold decrease in catalytic efficiency and an increase (approximately 50-fold) in Km Ru5P. For R173Q, a 15-fold diminution in Vmax and a 100-fold increase in Km Ru5P were observed. These observations implicate new components of the ribulose 5-phosphate binding site. Additionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well separated from other active site elements in the structure of the open form of the protein. Characterization of R186Q and R187Q mutants suggests that they influence the cooperativity of substrate binding.

Identification of catalytic residues in human mevalonate kinase.

cDNA encoding human mevalonate kinase has been overexpressed and the recombinant enzyme isolated. This stable enzyme is a dimer of 42-kDa subunits and exhibits a Vm = 37 units/mg, Km(ATP) = 74 microM, and Km(DL-MVA) = 24 microM. The sensitivity of enzyme to water-soluble carbodiimide modification of carboxyl groups prompted evaluation of four invariant acidic amino acids (Glu-19, Glu-193, Asp-204, and Glu-296) by site-directed mutagenesis. Elimination of Glu-19's carboxyl group (E19A, E19Q) destabilizes the enzyme, whereas E19D is stable but exhibits only approximately 2-fold changes in Vm and Km values. E296Q is a stable enzyme, which exhibits kinetic parameters comparable to those measured for wild-type enzyme. E193A is a labile protein, whereas E193Q is stable, exhibiting >50-fold diminution in Vm and elevated Km values for ATP (approximately 20-fold) and mevalonate (approximately 40-fold). Such effects would be compatible with a role for Glu-193 in interacting with the cation of the MgATP substrate. D204A and D204N are stable enzymes lacking substantial mevalonate kinase activity. The active sites of these Asp-204 mutants are intact, based on their ability to bind a spin-labeled ATP analog with stoichiometries and equilibrium binding constants that are comparable to those determined for wild-type enzyme. Competitive displacement experiments demonstrate that the Asp-204 mutants can bind ATP with Kd values that are comparable to estimates for wild-type enzyme. The >40,000-fold diminution in kcat for the Asp-204 mutants and the demonstration that they contain an otherwise intact active site support assignment of a crucial catalytic role to Asp-204. The assignment of Asp-204 as the catalytic base that facilitates deprotonation of the C-5 hydroxyl of mevalonic acid would be compatible with the experimental observations.

Evidence supporting a role for histidine-235 in cation binding to human 3-hydroxy-3-methyglutaryl-CoA lyase.

Histidine-235 of human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is the second basic residue in a conserved HXH motif. This residue is solvent accessible, readily reacting with the group specific reagent diethyl pyrocarbonate. Site-directed mutagenesis has been employed to substitute alanine or aspartate for H235. Characterization of the isolated H235A and H235D lyase mutants indicates that their tertiary structure is substantially intact. The mutant proteins, like the wild-type enzyme, are stoichiometrically modified by the affinity label, 2-butynoyl-CoA. Catalytic activity of the mutants is diminished by 15-fold and Km for HMG-CoA elevated approximately 4-fold in comparison with the values for wild-type enzyme. The function of H235 is suggested by investigation of the interaction of these enzymes with the dissociable divalent cation (e.g. Mg2+ or Mn2+) that is required for activity. ESR experiments show that wild-type enzyme forms a stable binary E*M complex. In contrast, H235A and H235D proteins do not efficiently form a binary complex. Significant interaction with cation (Mn2+) only occurs in the presence of the substrate analog, 3-hydroxyglutaryl-CoA. Similarly, when cation interaction is estimated in the presence of substrate using steady-state kinetic approaches, activator constants (Ka) and divalent cation Km values are measurable but are elevated by 15-90-fold over comparable estimates for the wild-type enzyme. The data confirm our earlier suggestion that both protein and substrate contribute ligands to HMG-CoA lyase's divalent cation activator. More specifically, the current observations suggest that H235 has an important function in cation binding.

Identification and functional characterization of an active-site lysine in mevalonate kinase.

We report the construction of an expression plasmid for rat mevalonate kinase and the overexpression of recombinant enzyme in Escherichia coli. The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa. The Michaelis constants (Km) for DL-potassium mevalonate (288 microM) and for ATP (1.24 mM) were in agreement with values reported for enzymes isolated from rat liver (Tanaka, R. D., Schafer, B. L., Lee, L. Y., Freudenberger, J. S., and Mosley, S. T. (1990) J. Biol. Chem. 265, 2391-2398). Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyridoxal phosphate (PLP). ATP (5 mM) afforded protection against inactivation, suggesting reaction of PLP with an active-site lysine. Mapping, isolation, and Edman degradation of the ATP-protectable peptide from [3H]PLP-inactivated borohydride-reduced mevalonate kinase allow assignment of lysine 13, a residue invariant in known mevalonate kinase sequences, as the modification site. These results represent the first identification of an active-site residue in mevalonate kinase. The function of lysine 13 was evaluated by replacing this residue with methionine. Vm of the mutant protein is diminished by 56-fold, suggesting that lysine 13 facilitates catalysis. Kd values of wild-type and mutant proteins for ATP were determined in electron spin resonance competition experiments. The observed 56-fold diminution in affinity for the mutant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.

Inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase and other Acyl-CoA-utilizing enzymes by 3-Oxobutylsulfoxyl-CoA.

3-Oxobutylsulfoxyl-CoA has been produced by oxidation of S-3-oxobutyl-CoA, the thioether analog of acetoacetyl-CoA. Avian hydroxymethylglutaryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded by the substrate, acetyl-CoA, suggesting that inactivation involves modification of the enzyme's active site. Pretreatment of HMG-CoA synthase with the inactivator blocks the enzyme's ability to form Michaelis and acetyl-S-enzyme intermediates, supporting the hypothesis that modification is active-site directed. Incubation of enzyme with oxobutylsulfoxyl-[32P]CoA followed by precipitation with trichloroacetic acid indicates that inactivation correlates with stoichiometric formation of a covalent adduct between enzyme and a portion of the inactivator that includes the CoA nucleotide. The observation of reagent partitioning suggests that HMG-CoA synthase catalyzes conversion of oxobutylsulfoxyl-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activity and reverses the covalent modification with release of CoASH. Oxobutylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a process that is also reversed by DTT. These three enzymes all contain active site cysteines, suggesting that inactivation results from disulfide formation between a cysteine and the CoA moiety of the inhibitor. The data are consistent with the hypothesis that enzymatic cleavage of oxobutylsulfoxyl-CoA results in the transient formation of a sulfenic acid derivative of CoA which subsequently reacts to form a stable disulfide linkage to protein.

Rhodobacter sphaeroides phosphoribulokinase: binary and ternary complexes with nucleotide substrate analogs and effectors.

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.

Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.

Evidence for the interaction of avian 3-hydroxy-3-methylglutaryl-CoA synthase histidine 264 with acetoacetyl-CoA.

Previous work on HMG-CoA synthase has implied the presence of a reactive active site histidine, prompting our examination of the possible function of invariant histidine residues by site-directed mutagenesis. Mutations encoding H197N, H264N/A, and H436N HMG-CoA synthases were constructed, and the mutant enzymes were overexpressed in Escherichia coli BL21(DE3). Kinetic characterization of the isolated synthase variants indicates that, while H197N and H436N enzymes behave similarly to wild-type synthase, H264N and H264A synthases exhibit significant differences. Although the k(m) for acetyl-CoA is not substantially altered, H264N/A synthases catalyze production of HMG-CoA at a diminished (approximately 25-fold slower) rate. In contrast, H264N/A synthases can efficiently catalyze the acetyl-CoA hydrolysis partial reaction exhibiting a k(m) for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme. These mutants also retain the ability to form significant levels of the acetyl-S-enzyme reaction intermediate. The functional catalysis of partial reactions argues that the H264 mutant proteins retain substantial structural integrity. In this context, it appears significant that the H264N/A synthases exhibit a approximately 100-fold increase in the k(m) for acetoacetyl-CoA. In order to test whether the two orders of magnitude effect may be largely attributed to a decreased affinity of acetoacetyl-CoA for these enzymes and, more specifically, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA's thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of acetoacetyl-CoA, was investigated. This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild-type synthase with an apparent Vmax that is approximately 100-fold lower and an apparent k(m) that is 25-fold higher than the values obtained using the physiological substrate, acetoacetyl-CoA. H264A synthase also catalyzes the turnover of S-(3-oxobutyl)-CoA; the diminution in rate supported by the alternative substrate is comparable in magnitude to the effect observed for wild-type enzyme. In contrast, H264A exhibits comparable apparent k(m) values for S-(3-oxobutyl)-CoA and acetoacetyl-CoA. Thus, unlike wild-type synthase, there is no penalty in terms of efficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate. These data suggest that the imidazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen of the thioester functionality.