Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis.

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue.

Affinity modification of E1-form of Na+, K+-ATPase revealed Asp-710 in the catalytic site.

An alkylating ATP analogue, gamma-[4-(N-2-chlorethyl-N-methylamino)]benzylamide ATP (C1RATP), covalently binds to the catalytic alpha-subunit of Na+, K+-ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Na+-form of the membrane-bound Na+, K+-ATPase modified with C1RATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4 degrees C, pH 1.5).