Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin.
Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248-257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (K(i)(app) values of 1 microM and 2.3 microM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/K(m, protoporhyrin IX)) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.
Shi
Zhen
Z
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA.
Ferreira
Gloria C
GC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D005294:Ferrochelatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D011487:Protein Conformation; D011524:Protoporphyrins / Q000494:pharmacology*
D011524:Protoporphyrins; C028842:N-methylprotoporphyrin IX; D005294:Ferrochelatase