Mutants, inhibitor-insensitive

Active Inhibitor-insensitive Kinase Mutants (Orthogonal Protein Kinases)... 

Figure 6.3 Schematic illustration of the generation of inhibitor-insensitive kinase mutants. The interaction of ATP-site competitors with kinase domains has been structurally characterized through the so-called Traxler model [10]. The part of the inhibitor that corresponds to the adenine ring binds to the hinge region of the kinase domain via H bonds. Next to the hinge region are the hydrophobic back pocket and the surface-exposed front pocket, which do not play a role in ATP binding. However, these pockets are extremely critical determinants in inhibitor binding, since the...

Figure 6.4 Inhibitor-insensitive EGF-R mutants and their use for target validation. The EGF-R signaling pathway is well understood. It is used here to prove the validity of the Axxima technology.

S-(2-Aminoethyl)-L-cysteine (AEC), H2N-CH2-CH2-S-CH2-CH(NH2)-COOH, a lysine analog, acts as a false feedback inhibitor on aspartokinase, which produces aspartylphosphate from aspartate. The inhibitor simulates, for aspartokinase, the absence of lysine and threonine, and as a consequence the AEC insensitive mutant is no longer inhibited by lysine and threonine. The result was a yield increase from 0 to 16 g L 1. 

An essential part of the rationale presented above under (a) consists of the identification of altered products of mitochondrial protein synthesis as a result of the mutation. Although this is not a sufficient criterion for mitochondrial specification (since an altered protein might arise as a result of a mutational alteration in a component of the mitochondrial protein-synthesizing machinery, i.e., one of the mt r- or tRNAs, (as in poky Neu-rospora), it is a necessary one. We have therefore devoted considerable effort to demonstrating the capability of the mitochondria in the mutant to perform some form of protein synthesis. We did this by showing that they were capable of incorporating (1) labeled formate into formylmethionyl-puromycin as a measure of mitochondrial polypeptide-chain initiations (see also next section) (2) labeled leucine into mitochondrial membrane proteins in a reaction that is insensitive to cycloheximide (CHX), but sensitive to chloramphenicol (CAP) and (3) that continued exposure of cells to the latter led to their conversion to petite phenocopies, s5 of characteristic aspects of their phenotype, such as the presence of cytochrome b, and that this change was reversed on removal of the inhibitor (see also Table I). 

Initially, the first and last enzymes of the lysine branch of the aspartate pathway illustrated in Fig. 1 were isolated from higher plants. The first enzyme, dihydrodipicolinate synthase (10), is sensitive to inhibition by the pathway product, lysine (Wallsgrove and Mazelis, 1981). Results obtained with extensively purified DHDP synthase from wheat suspension cultures indicate that lysine is a competitive inhibitor with respect to aspartate semialdehyde and a noncompetitive inhibitor with respect to pyruvate (Kumpaisal et ai, 1987). In vivo regulation of this enzyme by feedback inhibition is supported by the isolation of mutants which overproduce lysine and are characterized by a lysine-insensitive DHDP synthase (Negrutiu et ai, 1984). 

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