Kinetics of Regulatory Enzymes

The NAD-linked isocitrate dehydrogenase from baker's yeast was purified to homogeneity (as judged by gel filtration and polyacrylamide-gel electrophoresis) with an overall yield of 50% by using dilute solutions of the allosteric effector (AMP) to elute the enzyme specifically from CM-cellulose. This method preserves the allosteric properties of the crude enzyme. Although the pure enzyme shows only a single band on electrophoresis in the presence of sodium dodecyl sulphate, two types of subunit are observed in 8m-urea. The isoelectric point of the enzyme rises during purification, and this may reflect the partial loss of an additional low-molecular-weight component. Values are included for the amino acid composition and extinction coefficients of the pure enzyme.

Section: Vol 129 36supporting

confidence: 80%

“…and isocitrate. In partially purified preparations this activation occurs without change in the Hill coefficient of 4 for isocitrate, although the co-operativity of NADI binding is decreased by AMP (Atkinson et al, 1965). Atkinson and co-workers have recently purified the enzyme to homogeneity in the presence of citrate, which acts as a stabilizer.…”

mentioning

confidence: 99%

Purification of yeast isocitrate dehydrogenase

Illingworth

1972

“…However, in the presence of GMP a non-linear relationship is obtained and the lines curve upwards. These data, when replotted according to the Hill equation (Atkinson et al, 1965), give a Hill coefficient n= 1.0 in the absence of GMP, whereas in the presence of O.lmm-GMP and 0.2mM-GMP n = 1.15 and 1.34 respectively. The Hill coefficient n is considered to be a function of the number of substrate-binding sites on the enzyme molecule and of the degree of interaction between these sites (Atkinson et al, 1965).…”

Section: Resultsmentioning

confidence: 99%

“…These data, when replotted according to the Hill equation (Atkinson et al, 1965), give a Hill coefficient n= 1.0 in the absence of GMP, whereas in the presence of O.lmm-GMP and 0.2mM-GMP n = 1.15 and 1.34 respectively. The Hill coefficient n is considered to be a function of the number of substrate-binding sites on the enzyme molecule and of the degree of interaction between these sites (Atkinson et al, 1965). Values for the Hill coefficient greater than 1.0 indicate that the enzyme possesses regulatory or allosteric properties.…”

Section: Resultsmentioning

confidence: 99%

Inosine 5′-monophosphate dehydrogenase of Escherichia coli K12: the nature of the inhibition by guanosine 5′-monophosphate (Short Communication)

Lambden

Drabble

1973

When IMP is the variable substrate, IMP dehydrogenase (EC 1.2.1.14) gives non-linear Lineweaver-Burk plots in the presence of GMP. At 0.1mm-GMP the Hill coefficient n=1.15 and at 0.2mm-GMP n=1.34. In the absence of GMP n=1.0. The fact that n exceeds 1.0 in the presence of GMP indicates that the enzyme possesses regulatory (allosteric) properties. The inhibition by GMP is competitive with respect to IMP and non-competitive with respect to NAD(+).

“…The mechanism of action proposed by Atkinson, Hathaway & Smith (1965) for the yeast NADspecific isocitrate dehydrogenase appears to have considerable relevance to the characteristics of the pea NAD-specific isocitrate dehydrogenase. As discussed previously (Cox & Davies, 1969) the characteristics of binding of citrate and isocitrate to the latter suggest that both ligands are bound to the same sites.…”

mentioning

confidence: 96%

The effect of pH on the characteristics of the binding of nicotinamide–adenine dinucleotide by nicotinamide–adenine dinucleotide specific isocitrate dehydrogenase from pea mitochondria

Cox

Davies

1970

1. The effect of pH on the V(max.) and concentration of NAD(+) at half-maximum velocity at a constant isocitrate concentration was examined, and the results were related to the requirements for binding of H(+) ions to the enzyme. 2. The effect of varying the NAD(+) concentration on the pH optimum with constant isocitrate concentration was studied. 3. A comparison has been made between the effect of isocitrate concentration on the characteristics of binding of NAD(+) and the effect of NAD(+) concentration on the characteristics of isocitrate binding at three different pH values. 4. The mechanistic and metabolic significance of these studies is considered.