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.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: clinical course and description of causal mutations in two patients.

Hereditary deficiency of mitochondrial HMG-CoA synthase (mHS, OMIM 600234) is a poorly defined, treatable, probably underdiagnosed condition that can cause episodes of severe hypoketotic hypoglycemia. We present clinical follow-up and molecular analysis of the two known mHS-deficient patients. The diagnosis of mHS deficiency is challenging because the symptoms and metabolite pattern are not specific. Moreover, enzyme analysis is technically difficult and requires sampling of an expressing organ such as liver. The patients, now aged 16 and 6 y, have normal development and have had no further decompensations since diagnosis. Patient 1 is homozygous for a phenylalanine-to-leucine substitution at codon 174 (F174L). Interestingly, although the F174 residue is conserved in vertebrate mHS and cytoplasmic HS isozymes, a Leu residue is predicted in the corresponding position of HS-like sequences from Caenorhabditis elegans, Arabidopsis thaliana, and Brassica juncea. Bacterial expression of human F174L-mHS produces a low level of mHS polypeptide with no detectable activity. Similarly, in purified cytoplasmic HS, which in contrast to purified human mHS is stable and can be studied in detail, the corresponding F-->L substitution causes a 10,000-fold decrease in V(max) and a 5-fold reduction in thermal stability. Patient 2 is a genetic compound of a premature termination mutation, R424X, and an as-yet uncharacterized mutant allele that is distinguishable by intragenic single nucleotide polymorphisms that we describe. Molecular studies of mHS are useful in patients with a suggestive clinical presentation.

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.

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.

Role of His159 in yeast enolase catalysis.

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA. Metal binding studies support the above proposal. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281. Cha et al. (1994) J. Biol. Chem. 269, 2687-2694].

pH dependence of the reaction catalyzed by yeast Mg-enolase.

The pH dependence of the chemical shifts of the 31P resonances of enzyme-bound substrates 2-phosphoglycerate (PGA) and phosphoenolpyruvate (PEP) were measured to obtain further insight into the catalytic mechanism of yeast enolase. The 31P resonances of PGA and PEP bound to the enolase-Mg complex are individually observed by NMR. The Keq,internal = 1.5 favoring PEP was measured. A pH dependence of the 31P chemical shifts gives pKa values of 5.82 and 6.16 for bound PGA and PEP, respectively, indicating that both ligands bind predominantly with their phosphate groups as the dianionic species and their ionization has been altered. The phosphoryl group of PGA has been suggested as playing a role in catalysis [Nowak, T., Mildvan, A. S., and Kenyon, G. L. (1973) Biochemistry 12, 1690-1701]. The pH dependence of the kinetic parameters for Mg-enolase shows a single break in the plot of pKm, PGA vs pH at pH 6.27 with a pH independence above pH 7. This is consistent with the trianion of PGA preferably binding to the enzyme. The kcat profile gives pKA values of 5.94 and 8.35, and kcat/Km profiles give pKA values of 5.85, 6.25, and 8.39. Activation studies with Mg2+ show a pH independence for the activator constant (Ka), but a pH-dependent inhibition at higher concentrations of Mg2+. The log kcat and kcat/Ka profiles from Mg2+ activation give pKA values of about 5.9 and 8.4. These results confirm the importance of residues with pKA values of about 5.9 and 8.4 (His and Lys residues?) but do not support a function for the phosphoryl group of the substrate. The pH dependence of the Ki,Mg2+ gives pKA fits of 5. 95, 7.13, and 8.35. Data from cation inhibition suggest that the phosphate of the substrate and a His residue on enolase may bind the inhibitory Mg2+.