Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 Å, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.

Sample SK SK + 2 mM shikimate 93 The interaction of shikimate kinase from Envinia chrystanthemi with substrates

No abstract

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.

No abstract

Shikimate kinase was chosen as a convenient representative example of the subclass of a/b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 M urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJAEmol ) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other a/b domain proteins is discussed.