An investigation of an unusual histidyl residue in Escherichia coli B L-asparaginase through fluorescence quenching.

The tryptophanyl fluorescence of Escherichia coli B L-asparaginase is partially quenched by the protonated form of a base with pKa 6.0 at 25 degrees C, mu = 0.1. This base has been identified as a histidyl residue through the effect of ionic strength and solvent polarity on the pKa. In addition diethylpyrocarbonate which modifies two histidyl residues in the enzyme abolishes the fluorescenc titration and reduces enzymic activity by 90%. The temperature dependence of the histidine pKa is unusual, showing a minimum at 25 degrees C, a thermodynamic analysis of the data shows this to be due to a large negative delta Cp term associated with the ionisation. This is interpreted in terms of the movement of hydrophobic residues into the enzyme on deprotonation of the histidyl residue. The quantum yield of L-asparaginase and its temperature dependence have been measured. The quantum yield is high and there is a low activation energy for radiationless deactivation of the excited state both of which are consistent with a tryptophanyl environment remote from the solvent.