Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism.

An important sequence motif identified by sequence analysis is shared by the ACT domain family, which has been found in a number of diverse proteins. Most of the proteins containing the ACT domain seem to be involved in amino acid and purine synthesis and are in many cases allosteric enzymes with complex regulation enforced by the binding of ligands. Here we explore the current understanding of the ACT domain function including its role as an allosteric module in a selected group of enzymes. We will further describe in more detail three of the proteins where some understanding is available on function and structure: i) the archetypical ACT domain protein E. coli 3PGDH, which catalyzes the first step in the biosynthesis of L-Ser, ii) the bifunctional chorismate mutase/prephenate dehydratase (P-protein) from E. coli, which catalyzes the first two steps in the biosynthesis of L-Phe, and iii) the mammalian aromatic amino acid hydroxylases, with special emphasis on phenylalanine hydroxylase, which catalyzes the first step in the catabolic degradation of L-phenylalanine (L-Phe). The ACT domain is commonly involved in the binding of a small regulatory molecule, such as the amino acids L-Ser and L-Phe in the case of 3PGDH and P-protein, respectively. On the other hand, for PAH, and probably for other enzymes, this domain appears to have been incorporated as a handy, flexible small module with the potential to provide allosteric regulation via transmission of finely tuned conformational changes, not necessarily initiated by regulatory ligand binding at the domain itself.

The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type-II elastase.

Preparations of chymotrypsin from Atlantic cod are heterogeneous and previously gave rise to two active peaks when subjected to pH-gradient chromatography. Extension of the pH-gradient resolved a third protein peak with benzoyltyrosine ethylester hydrolytic activity. The first two peaks have been characterized as chymotrypsin variants and designated A and B, whereas the identity of the third peak was not clear. Analysis of this protein by Edman sequencing and mass spectrometry has now confirmed a high degree of identity with the predicted protein sequence from a recently described cDNA clone. That sequence was named elastase B by sequence comparison. As the present elastase deviates in 16 positions from that of elastase B, we have named it elastase C. The elastase C was active in hydrolysing typical substrates used by chymotrypsin, namely benzoyl-L-tyrosine ethylester and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, but inactive against the typical elastase substrates succinyl-Ala-Ala-Ala-p-nitroanilide and orcein-elastin. Comparison of the kinetic properties of the cod elastase C with bovine chymotrypsin and cod chymotrypsin variants A and B, using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, showed a lower catalytic efficiency of elastase C. The effects of several inhibitors on cod elastase C were identical to effects on chymotrypsins variants A and B, but dissimilar when compared with porcine pancreatic elastase. On the basis of the specificity and amino acid sequence, we conclude that the enzyme under study is most correctly classified as a type-II elastase.

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua.

The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsin variant B showed 72% sequence identity with the A-variant and 64% and 62%, respectively, with the bovine counterparts A and B, all consisting of 245 amino acids. This new sequence contains a higher proportion of charged residues compared with bovine chymotrypsin but fewer polar hydrogen-bond forming residues which might contribute to its lower thermostability. It also shares the emerging characteristics of other fish serine proteinases which have relatively higher methionine content, including a conserved Met-134 in a loop leading into a domain-connecting strand. The inherent mobility in methionine side-chains may contribute to the maintenance of flexibility at low temperatures. Several amino-acid sequence differences adjacent to the catalytic site are observed in the two cod chymotrypsin variants which also differ in kinetic properties. Unlike the mammalian chymotrypsins, which contain several autolysis sites, cod variant B only contains a single autolysis site. The three-dimensional structures of the A- and B-variants of cod has been modelled on the known crystal structure of bovine alpha-chymotrypsin showing almost superimposable structures.