Association-dissociation of transcarboxylase.

Transcarboxylase consists of a central subunit to which two sets of three subunits each are attached at opposite faces. Evidence obtained by various ultracentrifugal techniques has shown that there is an equilibrium between active forms of the enzyme with six, five, four, three, two, and one subunits attached to the central subunit. Since each attached subunit contains two biotins, the biotin content of these forms varies from 12 to 2. By reactive enzyme sedimentation at pH 5.5, it has been shown that the largest form of the enzyme (with 12 biotins and a molecular weight of 1.2 X 10(6) is active and that at pH 6.8 this form of the enzyme dissociates to an enzymatically active form containing three attached subunits. This result is in accord with previous observations (Wood, H.G., Chiao, J.P., and Poto, E.M.(1977), J Biol. Chem. 252, 1490; Wrigley, N.G., Chiao, J.P., and Wood, H.G. (1977), J Biol. Chem. 252, 1500) which showed that dissociation of three of the six attache subunits occurs preferentially from one face of the central subunit and that the remaining three attached subunits are quite firmly bound to the second face of the central subunit. Multiple peaks are obtained in sedimentation velocity experiments at 60 000 rpm because the rate of sedimentation outstrips the rate of equilibration of the different forms of transcarboxylase, whereas at 30 000 rpm a single peak of the multiple form of the enzyme is observed, since equilibration of the different forms keeps abreast of the sedimentation. The rate and extent of dissociation are increased by increase in temperature. It has been shown by centrifugation under oil that the association-dissociation of the large form of the enzymes with six, five, and four attached subunits is not affected by hydrostatic pressure. In contrast, the association-dissociation of the enzyme with three and two attached subunits appears to be affected by pressure. The accumulated results indicate that the native form of the enzyme in the cytosol of the cell consists of a series of enzymatically active forms with one to six attached subunits. The predominant forms in the cytosol will depend on the concentration of the constituent subunits, the pH, the concentration of ions (particularly divalent anions), and the temperature.

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.