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Nature Structural Biology Jul 2000Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human...
Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.
Topics: Alkaptonuria; Apoenzymes; Binding Sites; Catalysis; Crystallography, X-Ray; Dimerization; Dioxygenases; Homogentisate 1,2-Dioxygenase; Humans; Iron; Models, Molecular; Molecular Sequence Data; Oxygenases; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Structure-Activity Relationship
PubMed: 10876237
DOI: 10.1038/76756 -
Biochemical and Biophysical Research... Oct 1977
Topics: Apoenzymes; Cell Line; Humans; Isomerases; Kinetics; Methylmalonic Acid; Methylmalonyl-CoA Mutase; Mutation; Vitamin B 12
PubMed: 20894
DOI: 10.1016/0006-291x(77)90511-3 -
The Biochemical Journal Dec 1993The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been... (Comparative Study)
Comparative Study
The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been studied by following the substrate reaction continuously in presence of the metal ion or OP respectively. Although the native Zn(II)-containing and the Co(II)-reconstituted enzymes have closely similar Michaelis constants and maximal velocities, the kinetics for both the inactivation by OP and the reconstitution of the apoenzyme with the metal ions differs considerably. For Co(II), both the inactivation by OP and the reconstitution show simple kinetics, but for Zn(II), the inhibition by OP is a multi-phasic process [Wang, Wu, Wang, Zhou and Tsou (1992) Biochem. J. 281, 285-290], and the kinetics of reconstitution is also much more complicated. Both the native and the Co(II)-reconstituted enzymes are inhibited by excess of Zn(II), but not by Co(II). The inhibition by Zn(II) in excess and the reconstitution of the apoenzyme with Zn(II) are co-operative processes. The inhibition by Zn and its effect on the fluorescence emission of 1-anilinonaphthalene-8-sulphonic acid bound to the native enzyme indicate multiple Zn(II)-binding sites.
Topics: Amidohydrolases; Apoenzymes; Cobalt; Kinetics; Mathematics; Models, Theoretical; Phenanthrolines; Spectrometry, Fluorescence; Zinc
PubMed: 8257435
DOI: 10.1042/bj2960435 -
Acta Crystallographica. Section D,... Nov 2006The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors....
The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.
Topics: Apoenzymes; Crystallography, X-Ray; Escherichia coli; Protein Structure, Quaternary; Pyruvate Dehydrogenase Complex; Thiamine Pyrophosphate
PubMed: 17057342
DOI: 10.1107/S0907444906034408 -
Clinical Chemistry May 1986In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine...
In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activity, which is measured colorimetrically. The assay requires 100 microL of sample and covers the clinically significant range for TBG (less than 2.5 to 55 mg/L). The first result is obtained in 16 min; assay of 29 samples and their blanks is completed in less than 1 h. The lower limit of detection is about 2.5 mg/L. Between-assay CVs (n = 9) were less than 9%, within-assay CVs (n = 5) were less than 6%, and analytical recovery of TBG was 103-112%. Reagents are stable at 4 degrees C for at least five months. Results by this method for serum TBG (y) compared well with those determined by radioimmunoassay (x): y = 1.029x--0.352 (r = 0.990, n = 49, Syx = 1.165 mg/L). In addition, with 39 other sera the ratio of total thyroxin (by RIA) to TBG compared well with free thyroxin measured by equilibrium dialysis (r = 0.930) and the free thyroxin index (r = 0.970).
Topics: Apoenzymes; Glucose Oxidase; Humans; Immunoenzyme Techniques; Radioimmunoassay; Reagent Kits, Diagnostic; Thyroxine-Binding Proteins
PubMed: 3084130
DOI: No ID Found -
Proteins Sep 2021The main protease M , 3CL is an important target from coronaviruses. In spite of having 96% sequence identity among M from SARS-CoV-1 and SARS-CoV-2; the inhibitors used... (Comparative Study)
Comparative Study
The main protease M , 3CL is an important target from coronaviruses. In spite of having 96% sequence identity among M from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 M so far, were found to have differential inhibitory effect on M of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of M from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 M in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo M from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in M . Moreover, we have also investigated the network connectivity on the quaternary structure of M and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.
Topics: Apoenzymes; Binding Sites; Coronavirus 3C Proteases; Holoenzymes; Models, Molecular; Protease Inhibitors; Protein Multimerization; Protein Structure, Quaternary; Severe acute respiratory syndrome-related coronavirus; SARS-CoV-2
PubMed: 33983654
DOI: 10.1002/prot.26143 -
Biochemical and Biophysical Research... Mar 1961
Topics: Apoenzymes; Catechol Oxidase; Monophenol Monooxygenase; Oxidoreductases; Proteins
PubMed: 13751489
DOI: 10.1016/0006-291x(61)90240-6 -
The Journal of Biological Chemistry Jul 1965
Topics: Animals; Apoenzymes; Aspartate Aminotransferases; Carbon Isotopes; Chemical Phenomena; Chemistry; Hydrogen-Ion Concentration; Myocardium; Pyridoxal Phosphate; Pyridoxine; Research; Spectrophotometry; Swine; Vitamin B 6
PubMed: 14342324
DOI: No ID Found -
The Journal of Biological Chemistry Feb 1987In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim....
In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.
Topics: Alkaline Phosphatase; Animals; Apoenzymes; Apoproteins; Enzyme Activation; Liver Neoplasms, Experimental; Magnesium; Rats; Subcellular Fractions; Time Factors; Tissue Distribution; Zinc
PubMed: 3805040
DOI: No ID Found -
Biochemistry Jun 2005Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins and tetanus neurotoxin are the most potent toxins known to humans. Their potency coupled... (Comparative Study)
Comparative Study
Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins and tetanus neurotoxin are the most potent toxins known to humans. Their potency coupled with their specificity and selectivity underscores the importance in understanding their mechanism of action in order to develop a strategy for designing counter measures against them. To develop an effective vaccine against the toxin, it is imperative to achieve an inactive form of the protein which preserves the overall conformation and immunogenicity. Inactive mutants can be achieved either by targeting active site residues or by modifying the surface charges farther away from the active site. The latter affects the long-range forces such as electrostatic potentials in a subtle way without disturbing the structural integrity of the toxin causing some drastic changes in the activity/environment. Here we report structural and biochemical analysis on several mutations on Clostridium botulinum neurotoxin type E light chain with at least two producing dramatic effects: Glu335Gln causes the toxin to transform into a persistent apoenzyme devoid of zinc, and Tyr350Ala has no hydrolytic activity. The structural analysis of several mutants has led to a better understanding of the catalytic mechanism of this family of proteins. The residues forming the S1' subsite have been identified by comparing this structure with a thermolysin-inhibitor complex structure.
Topics: Alanine; Apoenzymes; Arginine; Asparagine; Binding Sites; Botulinum Toxins; Catalysis; Crystallization; Enzyme Activation; Glutamic Acid; Glutamine; Kinetics; Mutagenesis, Site-Directed; Neprilysin; Neurotoxins; Protease Inhibitors; Substrate Specificity; Thermolysin; Threonine; Tyrosine
PubMed: 15938619
DOI: 10.1021/bi050253a