scholarly journals Acylation of carcinogenic hydroxamic acids by carbamoyl phosphate to form reactive esters

1971 ◽  
Vol 124 (1) ◽  
pp. 69-74 ◽  
Author(s):  
P D. Lotlikar ◽  
L Luha
Keyword(s):  
Hydroxamic Acids ◽  
Phosphate Concentration ◽  
Acetyl Phosphate ◽  
Acylating Agent ◽  
Carbamoyl Phosphate ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Lag Period ◽  
Reactive N ◽  
Derivatives Of

1. Acylation of 2-(N-hydroxyacetamido)fluorene and several other aromatic hydroxamic acids by carbamoyl phosphate was studied and compared with the acylating activity of acetyl-CoA. Acetyl phosphate was also studied. 2. The carbamoylation reaction had a pH optimum of 4.5. The reaction had a lag period of 1h and was then linear for 4h. This linearity ranged between 0.5mm- and 8mm-carbamoyl phosphate concentration. 3. At pH7.5, acetyl-CoA was the most powerful acylating agent. Acetyl phosphate was a weaker acylating agent than either of the others. 4. Among the various hydroxamic acids tested with acetyl-CoA and carbamoyl phosphate at pH.7.5, 2-(N-hydroxyacetamido)fluorene was the most reactive. On the other hand the less reactive N-hydroxy derivatives of 2-acetamidonaphthalene, 2-acetamidophenanthrene and 4-acetamidostilbene reacted severalfold more with carbamoyl phosphate than with acetyl-CoA. 5. It is suggested that carbamoylation of aromatic hydroxamic acids might be one of the final activation steps in carcinogenesis by these compounds.

scholarly journals On the origin of biochemistry at an alkaline hydrothermal vent

2006 ◽  
Vol 362 (1486) ◽  
pp. 1887-1926 ◽  
Author(s):  
William Martin ◽  
Michael J Russell
Keyword(s):  
Hydrothermal Vent ◽  
Proton Gradient ◽  
Metabolic Energy ◽  
Acetyl Phosphate ◽  
Carbamoyl Phosphate ◽  
Acetyl Coa ◽  
Metabolic Systems ◽  
Acetyl Coa Pathway ◽  
Pyruvate Synthase ◽  
Carbonyl Sulphide

A model for the origin of biochemistry at an alkaline hydrothermal vent has been developed that focuses on the acetyl-CoA (Wood–Ljungdahl) pathway of CO 2 fixation and central intermediary metabolism leading to the synthesis of the constituents of purines and pyrimidines. The idea that acetogenesis and methanogenesis were the ancestral forms of energy metabolism among the first free-living eubacteria and archaebacteria, respectively, stands in the foreground. The synthesis of formyl pterins, which are essential intermediates of the Wood–Ljungdahl pathway and purine biosynthesis, is found to confront early metabolic systems with steep bioenergetic demands that would appear to link some, but not all, steps of CO 2 reduction to geochemical processes in or on the Earth's crust. Inorganically catalysed prebiotic analogues of the core biochemical reactions involved in pterin-dependent methyl synthesis of the modern acetyl-CoA pathway are considered. The following compounds appear as probable candidates for central involvement in prebiotic chemistry: metal sulphides, formate, carbon monoxide, methyl sulphide, acetate, formyl phosphate, carboxy phosphate, carbamate, carbamoyl phosphate, acetyl thioesters, acetyl phosphate, possibly carbonyl sulphide and eventually pterins. Carbon might have entered early metabolism via reactions hardly different from those in the modern Wood–Ljungdahl pathway, the pyruvate synthase reaction and the incomplete reverse citric acid cycle. The key energy-rich intermediates were perhaps acetyl thioesters, with acetyl phosphate possibly serving as the universal metabolic energy currency prior to the origin of genes. Nitrogen might have entered metabolism as geochemical NH 3 via two routes: the synthesis of carbamoyl phosphate and reductive transaminations of α-keto acids. Together with intermediates of methyl synthesis, these two routes of nitrogen assimilation would directly supply all intermediates of modern purine and pyrimidine biosynthesis. Thermodynamic considerations related to formyl pterin synthesis suggest that the ability to harness a naturally pre-existing proton gradient at the vent–ocean interface via an ATPase is older than the ability to generate a proton gradient with chemistry that is specified by genes.

scholarly journals Purification and Characterization of Two Extremely Thermostable Enzymes, Phosphate Acetyltransferase and Acetate Kinase, from the Hyperthermophilic Eubacterium Thermotoga maritima

1999 ◽  
Vol 181 (6) ◽  
pp. 1861-1867 ◽  
Author(s):  
Anne-Katrin Bock ◽  
Jürgen Glasemacher ◽  
Roland Schmidt ◽  
Peter Schönheit
Keyword(s):  
Molecular Mass ◽  
Thermotoga Maritima ◽  
Enzyme Reaction ◽  
Acetyl Phosphate ◽  
Temperature Optimum ◽  
Terminal Amino Acid ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Terminal Amino ◽  
Phosphate Acetyltransferase

ABSTRACT Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (α4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases fromClostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi ⇌ acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 μM, respectively; the apparentV max values (at 55°C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90°C and was not inactivated by heat upon incubation at 80°C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (α2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP ⇌ acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent V max values (at 50°C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90°C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4(1 M), which was most effective, the enzyme did not lose activity upon incubation at 100°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

scholarly journals Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2972-2985 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Rajesh G. Prabhu ◽  
Ying-Han Lin ◽  
Christine L. Miller ◽  
J. Seshu
Keyword(s):  
Borrelia Burgdorferi ◽  
Carbon Storage ◽  
Vertebrate Host ◽  
Acetyl Phosphate ◽  
Mobility Shift ◽  
Acetyl Coa ◽  
Site Specific ◽  
Content Type ◽  
Environmental Signals ◽  
Carbon Storage Regulator

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

scholarly journals Trends and Exceptions in the Interaction of Hydroxamic Acid Derivatives of Common Di- and Tripeptides with Some 3d and 4d Metal Ions in Aqueous Solution

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3941 ◽  
Author(s):  
Ozsváth ◽  
Bíró ◽  
Nagy ◽  
Buglyó ◽  
Sanna ◽  
...  
Keyword(s):  
Metal Ions ◽  
Hydroxamic Acid ◽  
Hydroxamic Acids ◽  
17O Nmr ◽  
Equilibrium Study ◽  
Sandwich Type ◽  
Uv Visible ◽  
First Time ◽  
Derivatives Of ◽  
Half Sandwich

By using various techniques (pH-potentiometry, UV-Visible spectrophotometry, 1H and 17O-NMR, EPR, ESI-MS), first time in the literature, solution equilibrium study has been performed on complexes of dipeptide and tripeptide hydroxamic acids—AlaAlaNHOH, AlaAlaN(Me)OH, AlaGlyGlyNHOH, and AlaGlyGlyN(Me)OH—with 4d metals: the essential Mo(VI) and two half-sandwich type cations, [(η6-p-cym)Ru(H2O)3]2+ as well as [(η5-Cp*)Rh(H2O)3]2+, the latter two having potential importance in cancer therapy. The tripeptide derivatives have also been studied with some biologically important 3d metals, such as Fe(III), Ni(II), Cu(II), and Zn(II), in order to compare these new results with the corresponding previously obtained ones on dipeptide hydroxamic acids. Based on the outcomes, the effects of the type of metal ions, the coordination number, the number and types of donor atoms, and their relative positions to each other on the complexation have been evaluated in the present work. We hope that these collected results might be used when a new peptide-based hydroxamic acid molecule is planned with some purpose, e.g. to develop a potential metalloenzyme inhibitor.

scholarly journals Acetyl-CoA:4-Hydroxybutinylbithiophene O-Acetyltransferase Isoenzymes from Tagetes patula Seedlings

1987 ◽  
Vol 42 (7-8) ◽  
pp. 885-890 ◽  
Author(s):  
Gernot Metschulat ◽  
Rainer Sütfeld
Keyword(s):  
Coenzyme A ◽  
Developmental Stages ◽  
Complete Separation ◽  
Tagetes Patula ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Acyltransferase Activity ◽  
Enzyme Preparations ◽  
Naturally Occurring ◽  
Acyl Coenzyme A

Naturally-occurring hydroxybutinylbithiophene derivatives were acylated by enzyme preparations of Tagetes patula seedlings in the presence of distinct acyl-Coenzyme A esters. The O-acyltransferase activity could only be detected after almost complete separation of the enzyme from counter-currently acting esterases which were present in the same extracts. This was achieved by affinity chromatography on Cibachron Blue A. During this procedure, the O-acyl-transferase was split, yielding two active fractions. Both had a Mr of 37,000 (±5,000), equal isoelectric properties, a pH optimum of pH 7.0, and were considerably inhibited in the presence of free Coenzyme A. Small differences existed in their affinities for their thiophenic substrates (3,4-dihydroxybutinylbithiophene and 4-hydroxybutinylbithiophene, respectively), as well as for various acyl-CoA esters as cosubstrates. With the preferred cosubstrate, acetyl-CoA, acylation took place at the 4-position of the butinyl side chain of the molecules, forming the naturally- occurring 4-acetoxybutinylbithiophene and 3-OH,4-OAc-butinylbithiophene, respectively. From the other acyl-CoA esters employed, only propionyl-CoA was likewise converted, forming the corresponding O-propionyl esters. The reactions observed are suggested to be catalyzed by two acetyl-CoA: 4-hydroxybutinylbithiophene O-acetyltransferase isoenzymes which exhibit different affinities for particular substrates and cosubstrates. The activities of both the isoenzymes changed drastically if plant material from different developmental stages was used as enzyme source. Therefore, it may be suggested that these isoenzymes play an important regulatory role in the metabolism of naturally-occurring hydroxy- and acetoxybutinylbithiophenes and their derivatives.

scholarly journals Pyruvate carboxylase catalysis of phosphate transfer between carbamoyl phosphate and ADP

1991 ◽  
Vol 273 (2) ◽  
pp. 443-448 ◽  
Author(s):  
P V Attwood ◽  
B D L A Graneri
Keyword(s):  
Active Site ◽  
Pyruvate Carboxylase ◽  
Reverse Reaction ◽  
Carbamoyl Phosphate ◽  
Acetyl Coa ◽  
Essential Requirement ◽  
Pyruvate Carboxylation ◽  
Ionizable Residues ◽  
Ph Profiles

In a reaction that is analogous to the phosphorylation of ADP from carboxyphosphate, pyruvate carboxylase catalyses the formation of ATP from carbamoyl phosphate and ADP at a rate that is about 0.3% of the pyruvate-carboxylation reaction and about 3% of the full reverse reaction. Acetyl-CoA stimulates the phosphorylation of ADP from carbamoyl phosphate but is not an essential requirement of the reaction. Mg2+ also stimulates the reaction, and in the range of Mg2+ concentrations considered the effect of V is much larger in the absence of acetyl-CoA than in its presence. Acetyl-CoA and Mg2+ may be acting in a co-operative way to stimulate the phosphorylation of ADP in a similar way to their effects on the pyruvate-carboxylation reaction. The phosphorylation of ADP by carbamoyl phosphate is also stimulated by the presence of biotin in the part of the active site where this reaction occurs, but again it is not absolutely required for the reaction to proceed. The pH profiles of the phosphorylation of ADP by carbamoyl phosphate indicate that there are at least two ionizable residues involved in the reaction, one of which probably has a role in the release of carbamate from the active site.

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