Proceso de la preparación y “misa luqt´aña” ‘entrega de la misa’.

The recent crystal structure determination of porphobilinogen

deam inase has show n that this enzyme has the same polypeptide-chain folds as proteins from two classes of binding proteins, the transferrins, in

particular, lactoferrin, and the group II periplasmic binding proteins (Louie, 1993). The structural similarities give rise to hypothesis concerning both the molecular evolution of these proteins and the mechanism by which

porphobilinogen deaminase catalyses its reaction.

The dom ains 1 and 2 of porphobilinogen deam inase resemble a num ber of binding proteins, which also have a core of tw o topologically similar domains. These binding proteins include the duplicated lobes of transferrins (lactoferrin and serum transferrin) and also the group II periplasmic receptors (the sulphate-, phosphate-, m altodextrin- and lysine / arginine / ornithine binding proteins) (Baker and Lindley, 1992; Quiocho, 1991). Of the 210 carbon atoms in a-helices in dom ains 1 and 2 of deaminase, 113 are found to be equivalent in the N-lobe of lactoferrin w hen the tw o structures are superimposed. Porphobilinogen deam inase and the binding proteins all share the same connectivity w ithin the two rearranged parallel a / p domains and have two interdom ain hinge segments. They also have similar lengths of secondary structural elements and similar overall tw ists of the core p sheets. The N -term inal dom ains of these proteins share the greatest structural similarity. How ever, there is no significant sequence identity (less than 12%) betw een porphobilinogen deam inase and the other proteins. The five residues E-N-R-A-D from porphobilinogen deam inase and the N-lobe of lactoferrin constitute the longest segm ent of identical sequence betw een deam inase and any of the binding proteins.

Chapter 1 : Introduction

A common feature of deaminase and binding proteins is the binding cleft form ed at the interface between the two domains, and the involvement of the amino term inus of one or more a-helices in forming the ligand binding site. For deaminase and several of the binding proteins the ligand is

oxyanionic. For example, the active site cleft of deam inase has to provide a binding site for up to twelve carboxylate groups carried on the side

substituents of the dipyrrom ethane and the polypyrrole product; in the transferrins, the initial step in iron ligation is the binding of the essential carbonate anion which provides two coordinate bonds for the ferric ion which is subsequently bound (Anderson et al., 1990).

Despite the lack of sequence homology among porphobilinogen deam inase and the binding proteins, there are several functionally im portant residues which occur at roughly equivalent positions. These include a serine (residue 129 in deaminase) that both caps the a-12 helix and hydrogen bonds to an acid oxygen on the ligand, an arginine (arg-131 in deaminase) near the amino term inus of this helix which hydrogen bonds to an acid oxygen on the ligand and an aspartate (residue 84 in deaminase) which has diverse roles such as ligand binding, catalysis and dom ain closure.

The interdom ain flexibility inherent in these proteins is also of considerable mechanistic importance to both the binding proteins and porphobilinogen deaminase. In a num ber of these proteins, both open and closed conformations have been observed crystallographically, the two conformations differing in the w idth of the interdom ain cleft (Rosenberg, 1991; Sack et al., 1989; Oh et al., 1993). Cleft w idening is brought about mainly from hinging about the two connecting interdom ain strands. In the N-lobe of apo-lactoferrin, exposed basic side chains in the open cleft m ight serve to attract the carbonate ion, in hololactoferrin a 54° relative rotation of the two dom ains closes the cleft around the bound ligand and assembles the

coordinate sphere for the ferric ion.

In accordance w ith the considerably larger size of the dipyrrom ethane cofactor and the tetrapyrrolic chain, the binding cleft in porphobilinogen deam inase has a larger volume than those in the binding proteins. In

deam inase, residues mainly from dom ain 1 form the catalytic and substrate binding sites, whereas residues primarily from dom ain 2 provide both the binding site for the cofactor and an internal cavity that m ight serve to

accommodate the growing chain. Therefore, a relative tw isting of the two dom ains provides a possible model for the mechanism by which the terminal ring of the growing chain is carried into the appropriate position to react with the next molecule of porphobilinogen.

To optimise conditions for substrate binding and catalysis, it is im portant in binding proteins to exclude solvent from the vicinity of the bound ligand in the closed binding cleft. An analogous function in

deaminase, to protect reactive intermediates, involves not only cleft closure but m ovem ent of the mobile lid. This lid, which is absent in binding proteins, is proposed to close over the active site cleft upon substrate binding (Louie, 1993; Louie, 1995).

The close structural resemblance amongst porphobilinogen deaminase and the binding proteins suggests that these proteins are related by divergent evolution (Louie, 1993). It can be speculated that porphobilinogen deaminase originated from a two dom ain binding protein specific for an anionic,

porphobilinogen-like or perhaps dipyrrolic ligand, and then acquired

catalytic groups and possibly the mobile lid. This ancestral porphobilinogen deam inase m ight have rudim entally catalysed the coupling of

porphobilinogen molecules within its binding cleft w ithout covalently attaching the first porphobilinogen to the polypeptide chain. The proposed evolutionary relationship is sum m arised below in figure 1.14.

Figure 1.14 The Proposed Evolutionary Relationship Among the Group I and Group II Periplasmic Binding Proteins. Porphobilinogen Deaminase and the Transferrins

Monomeric binding motif S tran d re a rra n g e m e n t D u p lica tio n o f d o m a in s A d d itio n of d o m a i n s Porphobilinogen Deaminase D u p lica tio n ol \ d o m a in s w ith a c q u isitio n o f a lo n g co n n ectin g se g m e n t trav ersin g

the e n tire seco n d d o m a in Group I I S tra n d I j re a rra n g e m e n t \ Group n Periplasmic Periplasmic Binding Binding

Chapter 1 : Introduction