Determinants of catalytic activity with the use of purified I, D and H subunits of the magnesium protoporphyrin IX chelatase from Synechocystis PCC6803

Abstract

The I, D and H subunits (ChlI, ChlD and ChlH respectively) of the magnesium protoporphyrin IX chelatase from Synechocystis have been purified to homogeneity as a result of the overexpression of the encoding genes in Escherichia coli and the production of large quantities of histidine-tagged proteins. These subunits have been used in an initial investigation of the biochemical and kinetic properties of the enzyme. The availability of pure ChlI, ChlD and ChlH has allowed us to estimate the relative concentrations of the three protein components required for optimal activity, and to investigate the dependence of chelatase activity on the concentrations of MgCl2, ATP and protoporphyrin IX. It was found that, whereas ChlD and ChlH are likely to be monomeric, ChlI can aggregate in an ATP-dependent manner, changing from a dimeric to an octameric structure. Subunit titration assays suggest an optimal ratio of ChlI, ChlD and ChlH of 2:1:4 respectively. However, the dependence of chelatase activity on increasing concentrations of ChlI and ChlH with respect to ChlD suggests that these two subunits, at least in vitro, 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 MgATP2-, the other as the chelated metal. The steady-state kinetic parameters were determined from continuous assays; the Km values for protoporphyrin, MgCl2 and ATP were 1.25 µM, 4.9 mM and 0.49 mM respectively. The rate dependence of Mg2+ was clearly sigmoidal with a Hill coefficient of 3, suggesting positive co-operativity. Initiating the reaction by the addition of one of the substrates in these continuous assays resulted in a significant lag period of at least 10 min before the linear production of Mg protoporphyrin. This lag was significantly decreased by preincubating ChlI and ChlD with ATP and MgCl2, and by mixing it with ChlH that had been preincubated with protoporphyrin IX, ATP and MgCl2. This suggests not only a close MgATP2--dependent interaction between ChlI and ChlD but also an interaction between ChlH and the protoporphyrin substrate that also is stimulated by ATP and MgCl2.