Biochemical comparison of the Neurospora crassa wild‐type and the temperature‐sensitive leucine‐auxotroph mutant leu‐5

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Abstract
The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The .**GRAPHIC**. value for leucine was lowered by high ATP concentrations and correspondingly the .**GRAPHIC**. was lowered by high leucine concentrations. The .**GRAPHIC**. was lowered by high pH, a pK value of 6.7 (at 30.degree. C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 .mu.M leucine, 0.3 .mu.M tRNALeu, and pH 7 the apparent Km values were .**GRAPHIC**. = 1,3 mM, .**GRAPHIC**. = 49 .mu.M and .**GRAPHIC**. = 0.15 .mu.M. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37.degree. C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28.degree. C, or grown at permissive temperature for 24 h followed by growth at 37.degree. C). We therefore think that the small difference in the Km value for the leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.