Purification and properties of histidinol dehydrogenase from Escherichia coli B

Histidinol dehydrogenase has been purified from a derepressed mutant of Escherichia coli B. A molecular weight of about 91,000 was estimated by gel filtration. The native enzyme seems to be composed of two similar subunits which have a molecular weight of 52,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pI of the enzyme as determined by isoelectric focusing is 4.75. The enzyme is maximally active at pH 9.5. It is highly specific for NAD+ and histidinol, with a Km (NAD+) of 0.57 mM and a Km (histidinol) of 14 microM. Mn2+ is required for maximal activity. The enzyme is completely inactivated by 8 M-urea but regains its activity very quickly upon removal of the urea. Mn2+ and histidinol protect the enzyme from heat inactivation.