Protein tyrosine kinases (PTKs1
) are key enzymes in cell signaling and play roles in a wide range of diseases including cancer (1
). Several PTKs, including Abl, are targeted clinically with therapeutic agents, and others are the subject of pharmaceutical development (3
). As members of the protein kinase superfamily, PTKs share conserved sequences and folds (2
), and yet they show distinct substrate selectivity (4
) and modes of regulation (6
). Evidence suggests that the mechanism of PTK-catalyzed phosphoryl transfer is dissociative, in which the bond to the leaving group ADP is largely broken prior to tyrosine phenol attack on the gamma phosphorus of ATP (7
). The alignment, orientation, and spacing of the tyrosine and ATP substrates by key active site residues are believed to be crucial in facilitating catalysis based on structural (8
), mutagenic (9
), and kinetic (10
The catalytic loop sequence, DLAARN, is conserved throughout the vast majority of the 90 human PTKs, but differs in the nine members of the Src family, which have the sequence DLRAAN () (2
). This relocation of the Arg from the D+4 position in most PTKs to the D+2 position in the Src family suggests an unusual functional plasticity. A crystal structure of an insulin receptor tyrosine kinase-bisubstrate analog complex is believed to capture the active conformation of the enzyme and reveals the orientation of the active site residues (8
). This structure shows that the side-chain of the catalytic loop Asp makes a hydrogen bond to the tyrosine hydroxyl and likely serves as the catalytic base. The catalytic loop Arg side-chain makes apparent hydrogen bonds to this Asp side-chain, as well as the substrate phenol oxygen. This triangle of hydrogen bonds () appears to provide a scaffold that aligns the reactants. It is assumed that the Src Arg residue, located in the non-canonical D+2 position, fulfills a similar role to that of the catalytic loop Arg in the D+4 position in non-Src PTKs.
Catalytic Loop of Selected Kinases
Mutagenic analysis of the catalytic loop Arg has been performed previously on PTK Csk (11
) and Src (12
). Mutation of the D+4 Arg in PTK Csk to Ala (R318A Csk) resulted in a dramatic kcat
drop of 3000-fold with relatively small (<10-fold) Km
changes. A Csk double mutant was prepared to mimic the Src-like catalytic loop (D+2 Arg Csk), and this mutant showed a 150-fold increased kinase activity compared with R318A Csk (11
). While still 20-fold less than wild type, the substantial kinase activity of D+2 Arg Csk mutant bolsters the argument for functional plasticity of the catalytic loop Arg residue.
In some cases, enzyme active site point mutants can be chemically rescued by small molecules that complement the deleted side-chains (13
). Indeed, chemical rescue of R318A Csk with a series of diamino compounds (guanidiniums, amidiniums, imidazoles) could restore substantial activity to this mutant (11
). Imidazole was the most efficient R318A Csk rescue agent, showed Km
= 20 mM, and stimulated kinase activity up to the level of D+2 Arg Csk. Related studies were carried out on Src, and it was shown that mutation of the D+2 Arg in Src to Ala (R388A Src) is 200-fold less active than wild type (12
). A Src double-mutant that mimics the canonical catalytic loop (D+4 Arg Src) was 2-fold more active than wild type. Imidazole rescued R388A Src to a level of 50% of wild type, with a Km
= 5 mM.
The ability to rescue R318A Csk and R388A Src to 5–50% of wild-type kcat
values with a non-toxic concentration of imidazole (< 50 mM) raised the interesting possibility that it might be feasible to complement these mutant tyrosine kinases in cells. Indeed, it was demonstrated that imidazole could restore Csk and Src functionality to mouse embryonic fibroblasts expressing replacement Arg → Ala mutant kinases (12
). Using this chemical rescue approach, new insights into the rapid signaling events have been obtained for Src and Csk (12
Although progress on the chemical rescue of mutant Src and Csk has been made, questions remain. Does the imidazole occupy an active site position mimicking the position of the catalytic loop Arg side-chain? Are Arg → Ala mutant PTKs beyond Src and Csk likely to show more efficient chemical rescue, like Src, or less efficient rescue, like Csk? Can alternative potent rescue combinations of kinase mutations and small molecules be identified? Is the imidazole interacting with the mutant kinase as the imidazolium or the neutral species? Here we use a combination of X-ray crystallography, and extended mutagenic and kinetic experiments on Abl and Src, to address these questions.