scholarly journals Wild-type and mutant d-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects

1995 ◽  
Vol 307 (1) ◽  
pp. 135-142 ◽  
Author(s):  
P B M van Bastelaere ◽  
H L M Kersters-Hilderson ◽  
A M Lambeir
Keyword(s):  
Kinetic Parameters ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Substrate Binding ◽  
Isotope Effects ◽  
Xylose Isomerase ◽  
Wild Type ◽  
Ion Dissociation ◽  
Solvent Isotope

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.

scholarly journals Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays

1998 ◽  
Vol 332 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Alejandro TOVAR-MÉNDEZ ◽  
Rogelio RODRÍGUEZ-SOTRES ◽  
Dulce M. LÓPEZ-VALENTÍN ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Free Enzyme ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Physiological Concentration

To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea maysleaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivois very low, but highly responsive to even small changes in the intracellular concentration of its substrate and effectors.

scholarly journals Manganese Homeostasis in Group A Streptococcus Is Critical for Resistance to Oxidative Stress and Virulence

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Andrew G. Turner ◽  
Cheryl-lynn Y. Ong ◽  
Christine M. Gillen ◽  
Mark R. Davies ◽  
Nicholas P. West ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Transition Metal ◽  
Metal Ion ◽  
Double Mutant ◽  
Efflux Pump ◽  
Group A Streptococcus ◽  
Wild Type ◽  
Group A

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448ΔmntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448ΔmntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448ΔmntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448ΔmntE-ΔperR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence. IMPORTANCE Manganese is traditionally viewed as a beneficial metal ion to bacteria, and it is also established that most bacteria can tolerate high concentrations of this transition metal. In this work, we show that in group A Streptococcus, mutation of the mntE locus, which encodes a transport protein of the cation diffusion facilitator (CDF) family, results in accumulation of manganese and sensitivity to this transition metal ion. The toxicity of manganese is indirect and is the result of a failure of the PerR regulator to respond to oxidative stress in the presence of high intracellular manganese concentrations. These results highlight the importance of MntE in manganese homeostasis and maintenance of an optimal manganese/iron ratio in GAS and the impact of manganese on resistance to oxidative stress and virulence.

Role of the Divalent Metal Ion in Sugar Binding, Ring Opening, and Isomerization by D-Xylose Isomerase: Replacement of a Catalytic Metal by an Amino Acid

Biochemistry ◽  
1994 ◽  
Vol 33 (6) ◽  
pp. 1488-1494 ◽  
Author(s):  
Karen N. Allen ◽  
Arnon Lavie ◽  
Arthur Glasfeld ◽  
Timothy N. Tanada ◽  
Daniel P. Gerrity ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ring Opening ◽  
Metal Ion ◽  
Xylose Isomerase ◽  
Divalent Metal ◽  
Sugar Binding ◽  
Divalent Metal Ion ◽  
Catalytic Metal

scholarly journals A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens

2002 ◽  
Vol 367 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Mario KLIMACEK ◽  
Bernd NIDETZKY
Keyword(s):  
Pseudomonas Fluorescens ◽  
Isotope Effects ◽  
Wild Type ◽  
Solvent Isotope Effect ◽  
General Base ◽  
Kinetic Isotope ◽  
Solvent Isotope ◽  
Auxiliary Role ◽  
Long Chain

Lys-295, Asn-300 and His-303 of d-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD+-dependent oxidation of d-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for d-fructose reduction by wild-type and mutants at pH10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11±1 on kcat for mannitol oxidation by K295A at pH(2H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa4-Asn-Xaa2-His (where ‘Xaa’ denotes ‘any amino acid').

scholarly journals Arthrobacter d-xylose isomerase: protein-engineered subunit interfaces

1993 ◽  
Vol 291 (2) ◽  
pp. 575-583 ◽  
Author(s):  
L Varsani ◽  
T Cui ◽  
M Rangarajan ◽  
B S Hartley ◽  
J Goldberg ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Ion ◽  
Thermal Inactivation ◽  
Xylose Isomerase ◽  
Crystallographic Analysis ◽  
Salt Bridges ◽  
Wild Type ◽  
Subunit Dissociation ◽  
Subunit Interface ◽  
A Subunit

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253→>Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

scholarly journals Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

1991 ◽  
Vol 275 (2) ◽  
pp. 447-452 ◽  
Author(s):  
M Lander ◽  
A R Pitt ◽  
P R Alefounder ◽  
D Bardy ◽  
C Abell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Enzyme Substrate ◽  
Arginine Residues

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

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