Catalytic mechanism of porphobilinogen synthase: the chemical step revisited by QM/MM calculations.

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C-C bond formation first mechanism and the C-N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism.

The first branching point in porphyrin biosynthesis: a systematic docking, molecular dynamics and quantum mechanical/molecular mechanical study of substrate binding and mechanism of uroporphyrinogen-III decarboxylase.

In humans, uroporphyrinogen decarboxylase is intimately involved in the synthesis of heme, where the decarboxylation of the uroporphyrinogen-III occurs in a single catalytic site. Several variants of the mechanistic proposal exist; however, the exact mechanism is still debated. Thus, using an ONIOM quantum mechanical/molecular mechanical approach, the mechanism by which uroporphyrinogen decarboxylase decarboxylates ring D of uroporphyrinogen-III has been investigated. From the study performed, it was found that both Arg37 and Arg50 are essential in the decarboxylation of ring D, where experimentally both have been shown to be critical to the catalytic behavior of the enzyme. Overall, the reaction was found to have a barrier of 10.3 kcal mol(-1) at 298.15 K. The rate-limiting step was found to be the initial proton transfer from Arg37 to the substrate before the decarboxylation. In addition, it has been found that several key interactions exist between the substrate carboxylate groups and backbone amides of various active site residues as well as several other functional groups.

Computational insights into the mechanism of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersubstrate C-N and C-C bonds. The active site consists of several invariant residues, including two lysyl residues (Lys210 and Lys263; yeast numbering) that bind the two substrate moieties as Schiff bases. Based on experimental studies, various reaction mechanisms have been proposed for this enzyme that generally can be classified according to whether the intersubstrate C-C or C-N bond is formed first. However, the detailed catalytic mechanism of PBGS remains unclear. In the present study, we have employed density functional theory methods in combination with chemical models of the two key lysyl residues and two substrate moieties in order to investigate various proposed reaction steps and gain insight into the mechanism of PBGS. Importantly, it is found that mechanisms in which the intersubstrate C-N bond is formed first have a rate-limiting barrier (17.5 kcal/mol) that is lower than those in which the intersubstrate C-C bond is formed first (22.8 kcal/mol).

Theoretical study of 5-aminolevulinic acid tautomerization: a novel self-catalyzed mechanism.

5-Aminolevulinic acid (5ALA) is the key synthetic building block in protoporphyrin IX (PpIX), the heme chromophore in mitochondria. In this study density functional theory calculations were performed on the tautomers of 5ALA and the tautomerization reaction mechanism from its enolic forms (5-amino-4-hydroxypent-3-enoic acid and 5-amino-4-hydroxypent-4-enoic acid) to the more stable 5ALA. The hydrated form 5-amino-4,4-dihydroxypentanoic acid was also studied. The lowest energy pathway of 5ALA tautomerization is by means of autocatalysis, in that an oxygen of the carboxylic group transfers the hydrogen atom as a "crane", with an activation energy of approximately 15 kcal/mol. This should be compared to the barriers of about 35 kcal/mol for water assisted tautomerization, and 60 kcal/mol for direct hydrogen transfer. For hydration of 5ALA, the water catalyzed activation barrier is found to be approximately 35 kcal/mol, approximately 5 kcal/mol lower than direct hydration.