Cooperativity in ligand binding to dihydrofolate reductase

Birdsall, B.; Burgen, A. S. V.; Miranda, J. Rodrigues de; Roberts, G. C. K.

doi:10.1021/bi00604a013

Cited by 48 publications

(85 citation statements)

References 18 publications

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“…This result implies that these compounds may interact with different but contiguous or partially overlapping subdomains of the same pharmacophore of an allosteric site. This finding is reminiscent of the simultaneous binding of 2,4-diaminopyrimidine and p-aminobenzoyl-glutamate (subcomponent moieties of methotrexate) to dihydrofolate reductase and their allosteric interactions with coenzyme analogs (Birdsall et al, 1978(Birdsall et al, , 1980. It has generally been assumed that muscarinic ligands that bind to the primary or, especially, allosteric site should have a basic nitrogen, although a few uncharged competitive antagonists ('carbo' analogs) have been described previously (Barlow and Tubby, 1974;Waelbroeck et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

2002

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WIN 51,pyrimido [1,2-a] 3 H]NMS dissociation from M 3 receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M 3 receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M 3 receptors that also supports positive cooperativity with ACh.

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Section: Discussionmentioning

confidence: 99%

Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

2002

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“…K,, is determined from the ratio of the Kd values for successive complexes, and is a measure of the interaction between these complexes. By convention, the Kd for the complex formed last is used as the numerator in these calculations, and a value for K,,,,<l indicates a negative interaction (Birdsall et al, 1978 and Calculated from k,,JKd, using the Kd for the L-G~u binary complex (l/K3) and the steady-state turnover number (kcat). Determined from the Kd for the L-G~u binary complex (l/K3) and the apparent second-order association rate constant determined…”

Section: L-glu Binding To the Gdh-nad' Complexmentioning

confidence: 99%

The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination

Basso

Engel

Walmsley

1993

European Journal of Biochemistry

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The binding of NAD' and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD' or the GDH-L-GIu binary complexes, but the route via the GDH-NAD' binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ mol-'. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ . mol-'.Glutamate dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate by ammonia to L-Glutamate, using nicotinamide coenzymes as follows:This reaction is central to the metabolism of both higher organisms, which need to dispose of excess nitrogen, and many lower organisms, which are able to assimilate nitrogen in the form of ammonia. Accordingly, GDH enzymes from a variety of sources have been isolated and studied (Goldin and Frieden, 1971 ;Smith et al., 1975). Mechanistically, the GDH studied in most detail is that from beef liver mitochondria. This enzyme has been studied using steady-state kinetic, transient kinetic and isotope equilibrium exchange methods in an attempt to elucidate the mechanism (e.g. Frieden, 1959a and b; Dalziel, 1969, 1970;Silverstein and Sulebele, 1973;Pantaloni and Iwatsubo, 1967;di Franco and Iwatsubo, 1972;Chen and Engel, 1975; Rife and Cleland, 1980;Fisher et al., 1987). However, an overall view of its mechanism is still elusive. With regard to the order of substrate binding, the only generally accepted fact is that GDH follow a sequential mechanism, in which a central complex exists with all the substrates simultaneously present. Analysis of the steady-state kinetics for oxidative deamination is often complicated by non-classical behaviour, usually attributed to allosteric interactions (Frieden, 1959a;Monod et al., 1963 ;Engel and Dalziel, 1969;Goldin and Frieden, 1971). In the case of beef GDH, saturation with the allosteric effector ADP simplifies the kinetic behaviour, The steady-state analysis also s...

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“…Fluorometric measurements of the binding of 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate to Lactobacillus casei cells revealed that the two fragments bind the bacterial enzyme simultaneously and cooperatively (5,6). Moreover, a recent study concerning the in vitro evaluation of the antimalarial activity of pyrimethamine and sulfadoxine against Plasmodium falciparum also suggested the simultaneous-inhibition hypothesis as a possible mechanism of potentiation (13).…”

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confidence: 96%

“…Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3), which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, has been regarded as one of the crucial targets for bacterial and protozoal chemotherapy. The enzyme was shown to be the molecular site of action of a number of antifolates (2,8,32).…”

mentioning

confidence: 99%

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

Sirawaraporn¹,

Yuthavong²

1986

Antimicrob Agents Chemother

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Dihydrofolate reductase was partially purified from a pyrimethamine-sensitive Plasmodium chabaudi clone and a pyrimethamine-resistant clone derived from it and used in a study of the inhibitory effect of pyrimethamine and sulfadoxine, both alone and in combination. Kinetic analysis of the inhibitory effect of sulfadoxine against the enzyme from pyrimethamine-sensitive and -resistant parasites revealed that the drug inhibited the former enzyme competitively, with an inhibition constant (K1.) of 0.7 + 0.4 mM, but inhibited the latter enzyme noncompetitively, with Ki, and K1i of 8.9 ± 1.2 and 4.1 ± 1.2 mM, respectively. Previous studies also showed competitive inhibition by pyrimethamine on the former enzyme and noncompetitive inhibition on the latter enzyme, with some 200-fold-lower affinity. Sulfadoxine and pyrimethamine exhibited a mutually potentiating effect on the enzyme activity, as revealed by the concave isoboles and the fractional inhibitions of less than unity. A potentiating effect was observed for the enzymes from both sources and was not dependent on the degree of the purification of the enzyme. Our results can be explained by assuming simultaneous binding of two inhibitors on the enzyme.

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Cooperativity in ligand binding to dihydrofolate reductase

Cited by 48 publications

References 18 publications

Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors

The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

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