Structural Dynamics and Catalytic Mechanism of Hydroxymethylbilane Synthase

Abstract

Hydroxymethylbilane synthase (HMBS), also known as porphobilinogen deaminase (HMBS; EC 2.5.1.61), is the third enzyme in the heme biosynthetic pathway. It catalyzes the stepwise polymerization of four molecules of porphobilinogen (PBG) into the linear tetrapyrrole, 1-hydroxymethylbilane (HMB). In humans, mutations of HMBS have been linked to acute intermittent porphyria (AIP). In the current thesis, classical and quantum mechanical calculations have been used to understand the structural dynamics and catalytic mechanism of HMBS. Molecular dynamics (MD) simulations of the E. coli HMBS through the different stages of pyrrole chain elongation suggested the importance of domain movements and the active site loop movement in the polymerization of four units of PBG. However, in the human HMBS (hHMBS), an additional 29-residue insert wedged between domains 1 and 3 prevents the domain motions. In hHMBS, residues R26, D99 and R167 are proposed to be important for the catalysis based on MD simulations and earlier hypothesis. The QM calculations were performed on a cluster model consisting of the active site of hHMBS enzyme. The addition of one molecule of PBG to the cofactor is carried out in four steps: (1) protonation of the PBG substrate; (2) deamination of PBG; (3) electrophilic addition of the deaminated substrate to the terminal pyrrole ring of the enzyme-bound cofactor and (4) deprotonation at the carbon atom at the ?-position of the penultimate ring. The rate limiting step for the complete mechanism was found to be the deamination of the PBG moiety. The QM/MM calculations demonstrated the significance of protein environment in obtaining accurate energies for the catalytic mechanism. The findings from this study provide a detailed understanding of the chain elongation mechanism using multi-scale modeling and would assist in future work aimed at modulating the activity of HMBS newline