scholarly journals Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5′-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide

1986 ◽  
Vol 236 (1) ◽  
pp. 193-200 ◽  
Author(s):  
J E Baggott ◽  
W H Vaughn ◽  
B B Hudson
Keyword(s):  
Adenosine Deaminase ◽  
Cytotoxic Effect ◽  
Competitive Inhibitor ◽  
Spectrophotometric Assay ◽  
Adenylate Deaminase ◽  
Catalysed Reaction ◽  
Progress Curve ◽  
Competitive Inhibitors ◽  
Methotrexate Treatment ◽  
Competitive Product

With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5′-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5′-adenylate deaminase respectively.

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses

1988 ◽  
Vol 233 (1273) ◽  
pp. 461-475 ◽  
Keyword(s):  
Channel Blocker ◽  
Competitive Inhibitor ◽  
Tissue Response ◽  
Receptor Density ◽  
Receptor Kinetics ◽  
Tissue Responses ◽  
Competitive Inhibitors ◽  
Transmitter Substance ◽  
Uncompetitive Inhibitor ◽  
Temporal Inhomogeneity

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist–receptor complex, A n R, has n = 1 ; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n > 1 and/or when tissue responses reflect local peak A n R, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e. g. a ‘channel blocker’) may be more effective against nerve-released agonist than against exogenous agonist.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

Phosphorylation of Coformycin and 2′ -Deoxycoformycin, and Substrate and Inhibitor Properties of the Nucleosides and Nucleotides in Several Enzyme Systems

1985 ◽  
Vol 40 (9-10) ◽  
pp. 710-714 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Piotr Lassota ◽  
David Shugara
Keyword(s):  
Nmr Spectroscopy ◽  
Adenosine Deaminase ◽  
Snake Venom ◽  
Purine Nucleoside Phosphorylase ◽  
H Nmr ◽  
Enzyme Systems ◽  
Adenylate Deaminase ◽  
Wheat Shoot ◽  
Neutral Media ◽  
Biological Aspects

Abstract Under conditions where 2′-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5′-phosphate in 15 - 20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of ≤ 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5′-phosphate in 20% overall yield. Coformycin-5′-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2′-deoxycoformycin-5′-phosphate, coformycin-5′-phosphate was a feeble substrate of snake venom 5′-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5′-AMP. With 5′-AMP analogues as substrate, the 5′-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values > 0.3 mᴍ. The 5′-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki > 0.2 mᴍ), but are potent inhibitors of adenylate deaminase {Ki ≤ 10−9 ᴍ). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5′-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.

scholarly journals Structural modeling of GSK3β implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Meenakshisundaram Balasubramaniam ◽  
Nirjal Mainali ◽  
Suresh Kuarm Bowroju ◽  
Paavan Atluri ◽  
Narsimha Reddy Penthala ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Three Dimensional ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Dimensional Structure ◽  
High Potency ◽  
Subsequent Formation ◽  
Inactive Conformation ◽  
Competitive Inhibitors ◽  
Physiological Pathways

Abstract Glycogen synthase kinase-3β (GSK3β) controls many physiological pathways, and is implicated in many diseases including Alzheimer’s and several cancers. GSK3β-mediated phosphorylation of target residues in microtubule-associated protein tau (MAPTAU) contributes to MAPTAU hyperphosphorylation and subsequent formation of neurofibrillary tangles. Inhibitors of GSK3β protect against Alzheimer’s disease and are therapeutic for several cancers. A thiadiazolidinone drug, TDZD-8, is a non-ATP-competitive inhibitor targeting GSK3β with demonstrated efficacy against multiple diseases. However, no experimental data or models define the binding mode of TDZD-8 with GSK3β, which chiefly reflects our lack of an established inactive conformation for this protein. Here, we used metadynamic simulation to predict the three-dimensional structure of the inactive conformation of GSK3β. Our model predicts that phosphorylation of GSK3β Serine9 would hasten the DFG-flip to an inactive state. Molecular docking and simulation predict the TDZD-8 binding conformation of GSK3β to be inactive, and are consistent with biochemical evidence for the TDZD-8–interacting residues of GSK3β. We also identified the pharmacophore and assessed binding efficacy of second-generation TDZD analogs (TDZD-10 and Tideglusib) that bind GSK3β as non-ATP-competitive inhibitors. Based on these results, the predicted inactive conformation of GSK3β can facilitate the identification of novel GSK3β inhibitors of high potency and specificity.

A KINETIC STUDY OF THE DEAMINATION OF SOME ADENOSINE ANALOGUES: SUBSTRATE SPECIFICITY OF ADENOSINE DEAMINASE

1966 ◽  
Vol 44 (3) ◽  
pp. 331-337 ◽  
Author(s):  
J. Lyndal York ◽  
G. A. LePage
Keyword(s):  
Substrate Specificity ◽  
Kinetic Study ◽  
Adenosine Deaminase ◽  
Competitive Inhibitor ◽  
Kinetic Constants ◽  
Glycosidic Linkage ◽  
Adenosine Analogues ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Marked Dependence

The kinetic constants Km and Vmax were determined for the deamination by adenosine deaminase of a series of analogues of adenosine containing "fraudulent" sugars. The configuration of the 2′-hydroxyl was found to be important for the binding of enzyme and substrate. The largest effect of changes in sugar structure was on the rate of breakdown of the enzyme–substrate complex to form products, i.e. Vmax. The nature of the configuration in the 3′-position was not important if the 2′-hydroxyl was trans to the glycosidic linkage; however, if the steric arrangement of the 2′-hydroxyl was cis to the glycosidic linkage, then Vmax showed a marked dependence on the nature of the 3′-substituent and its configuration. For instance, Vmax values were for arabinosyl adenine < 3′-deoxyarabinosyl adenine <lyxosyl adenine. 6-N-methyladenosine was found to be a competitive inhibitor of adenosine deaminase, with a Ki of 2 × 10−6M.

scholarly journals GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C β-Lactamase

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Jung-Hyun Na ◽  
Young Jun An ◽  
Sun-Shin Cha
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Phosphate Group ◽  
The Other ◽  
Extended Spectrum ◽  
Competitive Inhibitors ◽  
Class C

ABSTRACT Nucleotides were effective in inhibiting the class C β-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 μM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

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