1. The His-X-Asp motif is one of the most conserved structural components of pro
ID: 302651 • Letter: 1
Question
1. The His-X-Asp motif is one of the most conserved structural components of protein kinases. To uncover the functional role of the conserved His, a mutational analysis of Aurora A kinase was conducted. Following the kinetic analysis of various Aurora A kinase mutants, the following data was obtained: H254Y 61.4 ± 6.4 WT H254F 73.9 t 4.7 H254R 70.3 t 4.3 Km(ATP) (HM) 63.5+7.2 Use this data to answer the following questions. a. What mutations have been created? Explain the rationale for ONE of the changes. b. Specifically explain which parameter of enzyme kinetics has been affected by the mutational analysis. What does this suggest about the role of the His residue?Explanation / Answer
PARAMETER OF ENZYME KINETICS AFFECTED BY MUTATIONAL ANALYSIS
Carbohydrate-binding cleft of Bacillus licheniformis 1,3-1,4-?-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to ?-strand-(87–95) of the minor ?-sheet III of the protein core by a single disulfide bond at Cys61–Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. Mutants with a <2-fold increase inK mcorrespond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (k cat/K m) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with K m > 2-fold andk cat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in k cat), but it is slightly less stable. Many previously published studies were performed with recombinant GST-GK and it was assumed that the pure, GST free authentic enzymes would be functionally indistinguishable from the fusion. This assumption remained to be comprehensively tested. In the present investigation we therefore prepared pure GKs by cleaving off the N-terminal GST tag from wild type and 31 mutants.. This observation demonstrates that previously published biochemical genetic information and interpretations on "Glucokinase Disease" (almost entirely based on the analysis of GST tagged recombinant human glucokinase preparations) are biologically meaningful and that the present biophysical studies requiring pure enzyme in which the source of the TF signal is limited to the GK molecule are feasible.
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