Conclusions

The motivation for the development of this synthesis has been the proposed pathway for the biosynthesis of PBG (11).[36,37,49] In view of the results of our model studies we can interpret the postulated mechanism for the enzymatic formation of PBG (11). The foremost task of the enzyme would be to induce the crucial carbon-carbon bond formation (intermediate 69)(see figure 30).

Shemin has been the first to propose a mechanism for PBGS drawing a close analogy between PBGS and class I aldolases (see figure 31).[49,66] His postulated mechanism is mainly based on two arguments: The transformation has been formulated in strict analogy of PBGS with aldolases and the sequence of recognition is based on the observed formation of a compound which Shemin called a mixed pyrrole, whose proposed structure however was wrong as could be shown latter.[67]

Despite the knowledge of more than twenty gene derived protein sequences for PBGS from different sources, the sequence of events on the enzyme and the mechanistic details of the transformation are largely unknown still. The following experimental findings are relevant for the mechanism of PBGS (see figure 32).[36]

The substrate forming the propionic acid side chain is interacting first. At least one of the substrates is forming a covalent bond with the enzyme via a Schiff base. Most of the enzymes isolated so far need Zn2+ as an essential cofactor. To bind the Zn2+ the cysteines have to be in their reduced form. The enzyme is usually an homooctamer. The deprotonation leading from the pyrrolenine tautomer to the aromatic pyrrole is enantioselective and occurs therefore on the enzyme.[36,68]

To study the order in which the two substrate molecules bind to the enzyme, Jordan performed highly elegant single-turnover experiments.[69-71] Stoechiometric equivalents of labelled substrate and porphobilinogen synthase were rapidly mixed and after about a 100 ms added to a large excess of unlabelled substrate. The position of the radioactive label was determined by degradation. The pulse labelling could also be done using [5-13C] 5-aminolevulinic acid. The 13C-NMR spectrum of the product allowed to identify the position of the label directly.

Starting from his observations Jordan postulated an alternative mechanism for the formation of porphobilinogen (see figure 33).

Jordan postulates that after the formation of the Schiff base between the enzyme and the first substrate molecule, the second substrate molecule forms a new Schiff base to the enzyme bound 5-aminolevulinate. Only after this step follows the aldol reaction and the elimination which leads after deprotonation to the product.

At the same time Jordan postulated a mechanistic alternative (Jordan II),[36,68,72] which combines the events of recognition as indicated by the pulse labelling experiment with the mechanistic thinking of Shemin, which gives preference to the C-C-bond formation in the biosynthetic sequence (see figure 34).

Intensive studies using inhibitors which were specifically synthesized in order to test the mechanism of porphobilinogen synthase[73] as well as many other biochemical experiments especially concentrating on the influence of the different metal atoms on the reactivity have not allowed to distinguish unequivocally between the different proposed mechanisms for the biosynthesis of porphobilinogen.[36,74,75] Even the accumulation of over 20 gene derived protein sequences did not allow to decide which of the mechanistic alternatives is used by the enzyme. However due to the cloning and overexpressing of the porphobilinogen synthase large quantities of the enzyme became available. This has allowed to crystallize porphobilinogen synthase from different sources.[76] Very recently the successful crystallisation of porphobilinogen synthase and the structure determination with a resolution of 2 Å has been reported.[77,78]

The structure determination allows for the first time to have clear ideas about the active site and the quaternary structure of porphobilinogen deaminase. The structure clearly shows that the porphobilinogen deaminase is an octamer or better a tetramer of strongly interacting dimers. All active sites are directed towards the outside, so that 8 fully active sites are present. At the active site two lysines could be identified and the positions of the two Zn2+ ions has been located near to the active site as well. Another remarkable observation is the fact that the overall structure of porphobilinogen synthase resembles remarkably to the structure of aldolases. These combined observations leads the authors to postulate that the porphobilinogen synthase combines mechanistic and structural aspects of the two classes of aldolases: aldolase I and aldolase II. Therefore they give preference to a mechanism in which the C-C-bond formation is the crucial event of the biosynthetic pathway. It will be very exiting to see how the different approaches, analysis of the gene derived sequences, structural information from the X-ray diffraction data and inhibition results will help to unravel the mechanism of this beautiful enzyme.