Role of Metal Ions in Catalysis by Enolase:  An Ordered Kinetic Mechanism for a Single Substrate Enzyme † (original) (raw)

Modulatory Effects of Mg 2+ and Zn 2+ Ions on Monoesterase Activity of Wild- type and Mutant E. coli Alkaline Phosphatases

Ilorin Journal of Science, 2018

Metal cofactors and arginine-166 residue are active site participants in alkaline phosphatase catalysis. However, the mechanism by which the metal cofactors coordinate with arginine-166 residue during alkaline phosphatase catalysis is elusive. This study investigated the effects of Mg 2+ and Zn 2+ on monoesterase activity of wild-type and mutant E. coli alkaline phosphatases (ECAPs). The intact arginine-166 residue of wild-type ECAP was replaced by alanine and serine in the mutant ECAPs, R166A and R166S, respectively. Monoesterase activity of ECAP was measured by monitoring the rate of hydrolysis of para-nitrophenyl phosphate (pNPP). The monoesterase activity of wild-type ECAP was approximately 2-fold higher than the mutant ECAPs. Mg 2+ (0.1-10mM) increased the activities of wild-type and mutant enzymes in a concentration-dependent manner. Zn 2+ (0.05-0.1mM) slightly increased the activities of wild-type and mutants ECAPs. In the absence and in the presence of 10mM Mg 2+ or 0.1mM Zn 2+ , the maximum reaction rate of wild-type ECAP was higher than those of the mutant ECAPs while its Michaelis constant was lower than those of the mutant ECAPs. Findings in this study revealed that monoesterase activity of ECAP was greatly reduced by the loss of arginine-166 residue but its modulation by Mg 2+ and Zn 2+ ions was independent of arginine-166 residue.

Mechanism of enolase: the crystal structure of enolase-magnesium-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-.ANG. resolution

Biochemistry, 1991

Enolase in the presence of Mg2+ catalyzes the elimination of H 2 0 from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-MgZ+-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I / a ( l ) 2 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374.

Role of Metal Ions in the Reaction Catalyzed by l -Ribulose-5-phosphate 4-Epimerase †

Biochemistry, 2000

, and Y229F mutants of L-ribulose-5-phosphate 4-epimerase had 10, 1, and 0.1%, respectively, of the activity of the wild-type (WT) enzyme when activated by Zn 2+ , the physiological activator. Co 2+ and Mn 2+ replaced Zn 2+ in Y229F and WT enzymes, although less effectively with the His mutants, while Mg 2+ was a poorly bound, weak activator. None of the other eight tyrosines mutated to phenylalanine caused a major loss of activity. The near-UV CD spectra of all enzymes were nearly identical in the absence of metal ions and substrate, and addition of substrate without metal ion showed no effect. When both substrate and Zn 2+ were present, however, the positive band at 266 nm increased while the negative one at 290 nm decreased in ellipticity. The changes for the WT and Y229F enzymes were greater than for the two His mutants. With Co 2+ as the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absence of substrate and a weak absorption band at 508 nm. With substrate present, however, an intense absorption band at 555 nm ( ) 150-175) with a negative molar ellipticity approaching 2000 deg cm 2 dmol -1 appears with WT and Y229F enzymes. With the His mutants, the changes induced by substrate were smaller, with negative ellipticity only half as great. The WT, Y229F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydroxyacetone and glycolaldehyde phosphate with an initial k cat of 1.6 × 10 -3 s -1 . The initial rate slowed most rapidly with WT and H97N enzymes, which have the highest affinity for the ketopentose phosphates formed in the condensation. The EPR spectrum of enzyme with Mn 2+ exhibited a drastic decrease upon substrate addition, and by using H 2 17 O, it was determined that there were three waters in the coordination sphere of Mn 2+ in the absence of substrate. These data suggest that (1) the substrate coordinates to the enzyme-bound metal ion, (2) His95 and His97 are likely metal ion ligands, and Tyr229 is not a metal ion ligand, but may play another role in catalysis, possibly as an acid-base catalyst.

Effects of metal ions on the catalytic and thermodynamic properties of the aminopeptidase isolated from pronase

Journal of Inorganic Biochemistry, 1995

The effect of metal ions on the catalytic and thermodynamic properties of the aminopeptidase isolated from pronase has been investigated. A decrease in K M and enhanced activity were observed for most of the metal ions examined. Ca(II) exhibits the most prominent effect on enzyme activity. The observed stability constants for the enzyme-bound metal ions are in the range of 102-106 M-l, which is much smaller than that of a metalloenzyme, indicating that the metal ion is not an integral part of the enzyme. The complexation of L-leucine-p-nitroanilide and the transition metal ions causes a reduction in free substrate concentration and hence a concomitant decrease in enzyme activity. Therefore, care must be taken to account for this decrease in substrate concentration in order to obtain reliable kinetic parameters. Binding of E and S to form ES was accompanied by a decrease in Gibbs free energy, whereas a dramatic increase in the free energy was observed for the conversion of ES to ES*. Both the enthalpy and the entropy were found to be crucial in destabilizing ES*. In the presence of Ca(lI), ES is stabilized by ~ 1 kcal/mol and ES* by ~1.4 kcal/mol. The stabilization of ES by the presence of Ca(II) is reflected by a smaller KM value compared to that of the metal-free enzyme. The activation free energies for the process E + S ~ ES* were 10.8 and 9.4 kcal/mol for the metal-free and the Ca(II)-activated enzymes, respectively. The difference of ~ 1.4 kcal/mol in the activation free energy may account for enhanced activity by the presence of Ca(II).

Octahedral Coordination at the High Affinity Metal Site in Enolase; Crystallographic Analysis of the MG++-Enzyme from Yeast at 1.9 Angstroms Resolution

1995

The structure of the Mg2+ complex of yeast enolase has been determined from crystals grown in solutions of poly(ethy1ene glycol) at pH 8.1. Crystals belong to the space group P21 and have unit cell dimensions a = 72.5 A, b = 73.2 A, c = 89.1 A, and p = 104.4'. There is one dimer in the asymmetric unit. The current crystallographic R-factor is 19.0% for all recorded data to 1.9 8, resolution. The electron density indicates a hexacoordinate Mg2+ at the high-affinity cation binding site. The octahedral coordination sphere consists of a meridional arrangement of three carboxylate oxygens from the side chains of Asp 246, Asp 320, and Glu 295, and three well-ordered water molecules. Octahedral coordination is the preferred geometry for alkaline earth metal ions in complexes with oxygen donor groups. In previous crystallographic studies of enolase, Zn2+ and Mg2+ complexes at the high-affinity site were reported to exist in trigonal bipyramidal coordination. This geometry was suggested to enhance the electrophilicity of the metal ion and promote rapid ligand exchange [Lebioda, L., & Stec, B. (1989) J. Am. Chem. SOC. 11 1, 85 1 1-85 131. The octahedral arrangement of carboxylate and water ligands in the Mgn-enolase complex determined here is most consistent with reports of the Mn2+ and Mg2+ coordination complexes of mandelate racemase and muconate lactonizing enzyme. These latter enzymes have alp-barrel folds comparable to enolase. Furthermore, coordination to Mg2+ in the present study is compatible with the geometry observed in the enolase-(Mgn)2-phosphonoacetohydroxamate structure [Wedekind,

Kinetic and Magnetic Resonance Studies of the Role of Metal Ions in the Mechanism of Escherichia coli GDP-mannose Mannosyl Hydrolase, an Unusual Nudix Enzyme †

Biochemistry, 2002

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-R-D-sugars to yield the -D-sugar and GDP by nucleophilic substitution with inversion at the C1′ carbon of the sugar Biochemistry 39, 8603-8608]. GDPMH requires a divalent cation for activity such as Mn 2+ or Mg 2+ , which yield similar k cat values of 0.15 and 0.13 s -1 , respectively, at 22°C and pH 7.5. Kinetic analysis of the Mn 2+ -activated enzyme yielded a K m of free Mn 2+ of 3.9 ( 1.3 mM when extrapolated to zero substrate concentration (K a Mn 2+ ), which tightened to 0.32 ( 0.18 mM when extrapolated to infinite substrate concentration (K m Mn 2+ ). Similarly, the K m of the substrate extrapolated to zero Mn 2+ concentration (K S GDPmann ) 1.9 ( 0.5 mM) and to infinite Mn 2+ concentration (K m GDPmann ) 0.16 ( 0.09 mM) showed an order of magnitude decrease at saturating Mn 2+ . Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn 2+ binding studies, monitoring the concentration of free Mn 2+ by EPR and of bound Mn 2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water protons (PRR), detected three Mn 2+ binding sites per enzyme monomer with an average dissociation constant (K D ) of 3.2 ( 1.0 mM, in agreement with the kinetically determined K a Mn 2+ . The enhancement factor ( b ) of 11.5 ( 1.2 indicates solvent access to the enzyme-bound Mn 2+ ions. No cross relaxation was detected among the three bound Mn 2+ ions, suggesting them to be separated by at least 10 Å. Such studies also yielded a weak dissociation constant for the binary Mn 2+ -GDP-mannose complex (K 1 ) 6.5 ( 1.0 mM) which significantly exceeded the kinetically determined K m values of Mn 2+ , indicating the true substrate to be GDP-mannose rather than its Mn 2+ complex. Substrate binding monitored by changes in 1 H-15 N HSQC spectra yielded a dissociation constant for the binary E-GDPmannose complex (K S GDPmann ) of 4.0 ( 0.5 mM, comparable to the kinetically determined K S value (1.9 ( 0.5 mM). To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K I ) 46 ( 27 µM), was studied. Binding studies revealed a weak, binary E-GDP complex (K D GDP ) 9.4 ( 3.2 mM) which tightened ∼500-fold in the presence of Mn 2+ to yield a ternary E-Mn 2+ -GDP complex with a dissociation constant, K 3 GDP ) 18 ( 9 µM, which overlaps with the K I GDP . The tight binding of Mn 2+ to 0.7 ( 0.2 site per enzyme subunit in the ternary E-Mn 2+ -GDP complex (K A ′ ) 15 µM) and the tight binding of GDP to 0.8 ( 0.1 site per enzyme subunit in the ternary E-Mg 2+ -GDP complex (K 3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn 2+ -GDP complex ( T ) 4.9 ( 0.4) indicates decreased solvent access to the active site Mn 2+ , consistent with an E-Mn 2+ -GDP bridge complex. Fermi contact splitting (4.3 ( 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an inner sphere E-Mn 2+ -GDP complex. The number of water molecules coordinated to Mn 2+ in this ternary complex was determined by ESEEM studies in D 2 O to be two fewer than on the average Mn 2+ in the binary E-Mn 2+ complexes, consistent with bidentate coordination of enzyme-bound Mn 2+ by GDP. Kinetic, metal binding, and GDP binding studies with Mg 2+ yielded dissociation constants similar to those found with Mn 2+ . Hence, GDPMH requires one divalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike its homologues the MutT pyrophosphohydrolase, which requires two, or Ap 4 A pyrophosphatase, which requires three.

Metal Ions Stabilize a Dimeric Molten Globule State between the Open and Closed Forms of Malic Enzyme

Biophysical Journal, 2007

Malic enzyme is a tetrameric protein with double dimer quaternary structure. In 3-5 M urea, the pigeon cytosolic NADP 1-dependent malic enzyme unfolded and aggregated into various forms with dimers as the basic unit. Under the same denaturing conditions but in the presence of 4 mM Mn 21 , the enzyme existed exclusively as a molten globule dimer in solution. Similar to pigeon enzyme (Chang, G. G., T. M. Huang, and T. C. Chang. 1988. Biochem. J. 254:123-130), the human mitochondrial NAD 1-dependent malic enzyme also underwent a reversible tetramer-dimer-monomer quaternary structural change in an acidic pH environment, which resulted in a molten globule state that is also prone to aggregate. The aggregation of pigeon enzyme was attributable to Trp-572 side chain. Mutation of Trp-572 to Phe, His, Ile, Ser, or Ala abolished the protective effect of the metal ions. The cytosolic malic enzyme was completely digested within 2 h by trypsin. In the presence of Mn 21 , a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with M r of 37 kDa, and this polypeptide was resistant to further digestion. These results indicate that, during the catalytic process of malic enzyme, binding metal ion induces a conformational change within the enzyme from the open form to an intermediate form, which upon binding of L-malate, transforms further into a catalytically competent closed form.