Sulfhydryl inhibitors and enzyme-substrate interactions of carbamyl phosphate synthetase

Abstract

1. 1. The indispensable cofactor for carbamyl phosphate synthetase (ATP: carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5), N- acetyl- l-glutamate , markedly accelerates inactivation of this enzyme by the disulfide 5,5′-dithiobis(2,2′-nitrobenzoate) (DTNB). This effect has been used to examine the interaction of the enzyme with its different substrates and cofactors. The following results have been obtained. 2. 2. ATP in the presence of Mg 2+ is the only substrate to protect against the inactivation. This protection is considerably enhanced if K 2+ is present. HCO 3 − augments the protective effect of ATP and K +, but provides no protection by itself. ADP does not protect against the inactivation. 3. 3. Mg 2+ has been found to increased the rate of inactivation. This effect is particularly marked when acetyl glutamate is present. In the absence of Mg 2+, the enzyme appears to bind acetyl glutamate at two sites, the second of which is detectable only at high concentrations of acetyl glutamate. Furthermore, inactivation is much slower at all except high levels of cofactor. In contrast, when Mg 2+ is present, the rate of inactivation is much greater, and the second binding site for acetyl glutamate is not detectable. 4. 4. The significance of these findings for the enzymatic reaction has been tested by determining the K m for acetyl glutamate in solutions containing either a slight excess of ATP over Mg 2+, or a large excess of Mg 2+ over ATP. When unbound Mg 2+ is low, the K m for acetyl glutamate is doubled in value, and the highest levels of acetyl glutamate produce an inhibition of the reaction. When the level of free Mg 2+ is high, there is a lower K m for acetyl glutamate, and no inhibition by acetyl glutamate. If the inhibitory effects of high acetyl glutamate concentration in the plot with low Mg 2+ concentration are ignored, the extrapolated maximum velocity is identical in both cases. 5. 5. None of the other substrates of the enzyme produce any significant effect upon the inactivation. 6. 6. The inactivation of the enzyme by organic mercurials, which is reduced instead of increased by acetyl glutamate, has been reinvestigate in the light of the above results. Mg 2+ reduces the protection by acetyl glutamate, but does not reduce inactivation in the absence of cofractor. Comparison of protection by acetyl glutamate against a variety of organic mercurials has revealed that the cofractor protects against mercurials in a manner which is independent of the nature of the organic portion of the inhibitor.