Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS

2011 ◽  
Vol 435 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Apirat Chaikuad ◽  
Naeem Shafqat ◽  
Ruby Al-Mokhtar ◽  
Gus Cameron ◽  
Anthony R. Clarke ◽  
...  
Keyword(s):  
Hormonal Contraception ◽  
Anion Recognition ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Human Sperm ◽  
Glycolytic Enzyme ◽  
Soluble Form ◽  
Active Centre ◽  
Recognition Sites

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN–NAD+–phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN–NAD+–glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

scholarly journals Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

2015 ◽  
Vol 81 (7) ◽  
pp. 2423-2432 ◽  
Author(s):  
M. Taillefer ◽  
T. Rydzak ◽  
D. B. Levin ◽  
I. J. Oresnik ◽  
R. Sparling
Keyword(s):  
Malate Dehydrogenase ◽  
Malic Enzyme ◽  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Cellulosic Biomass ◽  
Content Type ◽  
Malic Enzyme Activity

ABSTRACTClostridium thermocellumproduces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role inC. thermocellummetabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with akcatof 45.8 s−1or 14.9 s−1, respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with akcatof 520.8 s−1. The malic enzyme used NADP+as a cofactor along with NH4+and Mn2+as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with aKiof 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4+based on the characterization of the malate dehydrogenase and malic enzyme.

scholarly journals Characterization of recombinant fructose-1,6-bisphosphatase gene mutations: evidence of inhibition/activation of FBPase protein by gene mutation

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Gemma Topaz ◽  
Victor Epiter-Smith ◽  
Cristina Robalo ◽  
Megan Emad ◽  
Vanessa Ford ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Gene Mutations ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Coding Region ◽  
Functional Relationships ◽  
Fructose 1,6 Bisphosphatase ◽  
Natural Inhibitors

Abstract Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants (Ki’s) for natural inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP) compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance—and medical relevance—of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.

scholarly journals Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

2000 ◽  
Vol 66 (6) ◽  
pp. 2484-2490 ◽  
Author(s):  
Hak Soo Seo ◽  
Yeon Jong Koo ◽  
Jae Yun Lim ◽  
Jong Tae Song ◽  
Chung Ho Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Sequential Reaction ◽  
E Coli ◽  
Fusion Enzyme ◽  
Bifunctional Fusion Enzyme ◽  
Sequential Reactions

ABSTRACT To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The Km values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, thek cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k cat/Km ). The Km and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-d-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

scholarly journals An Electrochemical Approach to Follow and Evaluate the Kinetic Catalysis of Ricin on hsDNA

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 405
Author(s):  
George Oliveira ◽  
José Maurício Schneedorf
Keyword(s):  
Catalytic Efficiency ◽  
Ricin Toxin ◽  
Wave Voltammetry ◽  
Progress Curve ◽  
Electrochemical Approach ◽  
Electro Oxidation ◽  
Castor Seeds ◽  
Identification And Characterization ◽  
Medical Countermeasures

International authorities classify the ricin toxin, present in castor seeds, as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin are considered the first actions for its risk assessment during a suspected exposure, parallel to the development of therapeutic and medical countermeasures. In this study, we report the kinetic analysis of electro-oxidation of adenine released from hsDNA by the catalytic action of ricin by square wave voltammetry. The results suggest that ricin-mediated adenine release exhibited an unusual kinetic profile, with a progress curve controlled by the accumulation of the product and the values of the kinetic constants of 46.6 µM for Km and 2000 min−1 for kcat, leading to a catalytic efficiency of 7.1 × 105 s−1 M−1.

scholarly journals Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

1992 ◽  
Vol 282 (3) ◽  
pp. 711-714 ◽  
Author(s):  
E Blée ◽  
F Schuber
Keyword(s):  
Glycine Max ◽  
Gel Filtration ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Epoxide Hydrolases ◽  
Major Activity ◽  
Apparent Homogeneity ◽  
A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

scholarly journals Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

2005 ◽  
Vol 71 (3) ◽  
pp. 1581-1590 ◽  
Author(s):  
Luguang Wu ◽  
Robert G. Birch
Keyword(s):  
Catalytic Efficiency ◽  
High Ratio ◽  
Enzyme Active Site ◽  
E Coli ◽  
Pantoea Dispersa ◽  
Temperature Ranges ◽  
Lower Ratio ◽  
High Sucrose ◽  
Sucrose Isomerase

ABSTRACT Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the V max was 638 U mg−1, and the K cat/Km was 1.79 × 104 M−1 s−1, compared to a Km of 76.0 mM, a V max of 423 U mg−1, and a K cat/Km of 0.62 × 104 M−1 s−1 for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

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