ISSN:
1573-4919
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Summary Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryoprotectants. Their diverse chemical structure and the normal stoichiometry and pattern of positional isotopic exchange for the reaction catalyzed in their presence strongly supports an allosteric mechanism for activation by the physiological activator, acetylglutamate, and by the cryoprotectants. Activation appears not to require any specific group in the cryoprotectants, but different agents activate to different degrees. All cryoprotectants found to activate are good hydrogen-bond formers and have low molecular weight (〈1 000 for polyethylene glycols). This suggests that the cryoprotectant must associate with the enzyme to activate. In the presence of acetylglutamate all cryoprotectants tested, irrespective of their capacity to activate, inhibit. Carbamoyl phosphate synthetase I has activity in the absence of cryoprotectants and acetylglutamate. Under these conditions the Km's for ATP and K+ are very high, the Km for NH4 + is very low, the Vmax appears not to exceed 15% of that at saturation of acetylglutamate and the ratio of ATPase to carbamoyl phosphate synthetase activity is ca. three times that of the acetylglutamate activated enzyme. Acetylglutamate lowers the Km's for ATP and K+, and increases the Km for NH4 + and the Vmax. From steady state kinetics, pulse-chase, and measurements of the rate of activation, an allosteric model for activation of the enzyme by acetylglutamate is proposed in which there are two active species, R and R - AG (AG = acetylglutamate), which have similar but not identical activities. One molecule of ATP binds with high affinity to active and inactive enzyme species, but K+ and the other molecule of ATP bind preferentially to the active species. In the absence of acetylglutamate, glycerol (an effective activator) reduces the Km's for ATP and K+, increases the Vmax, and dramatically reduces the S0.5 for acetylglutamate to 〈1 μM for the synthetase reaction. In the presence of excess acetylglutamate, glycerol markedly reduces the Km for ATP for the synthetase, decreases the Vmax, NH4 + becomes a more effective activator than K+, and Na+ activates appreciably. From these and other kinetic measurements on the ATPase activity, the partial reverse reaction and rates of activation, and from pulse chase observations, the effect of glycerol is rationalized in terms of the allosteric model proposed for the actions of acetylglutamate if: a) glycerol reduces the rate of transformation and alters the allosteric equilibria between active and inactive enzyme species, and b) glycerol inhibits the active enzyme, at least in part due to slower conformational changes associated with dissociation of the products and binding of substrates. Glycerol also activates carbamoyl phosphate synthetase from E. coli in the absence of ornithine. This and the above findings are compared with data on the actions of cryoprotectants on carbamoyl phosphate synthetase II (EC 6.3.5.5) from rat liver. There are considerable similarities in the actions of these agents on the three enzymes. The relationship between these findings and the actions of cryoprotectants on other enzymes and possible physiological implications are discussed.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00225260
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