DETERMINANTS OF CATALYTIC ACTIVITY WITH THE USE OF PURIFIED I-SUBUNITS, D-SUBUNITS AND H-SUBUNITS OF THE MAGNESIUM PROTOPORPHYRIN-IX CHELATASE FROM SYNECHOCYSTIS PCC6803

The I, D and H subunits (ChlI, ChlD and ChlH respectively) of the magn esium protoporphyrin IX chelatase from Synechocystis have been purifie d to homogeneity as a result of the overexpression of the encoding gen es in Escherichia coli and the production of large quantities of histi dine-tagged proteins. These subunits have been used in an initial inve stigation of the biochemical and kinetic properties of the enzyme. The availability of pure ChlI, ChlD and ChlH has allowed us to estimate t he relative concentrations of the three protein components required fo r optimal activity, and to investigate the dependence of chelatase act ivity on the concentrations of MgCl2, ATP and protoporphyrin IX. It wa s found that, whereas ChlD and Chili are likely to be monomeric, ChlI can aggregate in an ATP-dependent manner, changing from a dimeric to a n octameric structure. Subunit titration assays suggest an optimal rat io of ChlI, ChlD and ChlH of 2:1:4 respectively. However, the dependen ce of chelatase activity on increasing concentrations of ChlI and ChlH with respect to ChlD suggests that these two subunits, at least in vi tro, behave as substrates in their interaction with ChlD. Mg chelation could not be detected unless the Mg2+ concentration exceeded the ATP concentration, suggesting at least two requirements for Mg2+, one as a component of MgATP(2-), the other as the chelated metal. The steady-s tate kinetic parameters were determined from continuous assays; the K- m values for protoporphyrin, MgCl2 and ATP were 1.25 mu M, 4.9 mM and 0.49 mM respectively. The rate dependence of Mg2+ was clearly sigmoida l with a Hill coefficient of 3, suggesting positive co-operativity. In itiating the reaction by the addition of one of the substrates in thes e continuous assays resulted in a significant lag period of at least 1 0 min before the linear production of Mg protoporphyrin. This lag was significantly decreased by preincubating ChlI and ChlD with ATP and Mg Cl2 and by mixing it with ChlH that had been preincubated with protopo rphyrin IX, ATP and MgCl2. This suggests not only a close MgATP(2-)-de pendent interaction between ChlI and ChlD but also an interaction betw een ChlH and the protoporphyrin substrate that also is stimulated by A TP and MgCl2.