Diphtheria toxin (DT) catalyzes the transfer of ATP-ribose from NAD to elongation factor 2 thus inhibiting protein synthesis and ultimately resulting in cell death. It has be proposed that the histidine at position 21 of the DT A subunit plays an important role in the ADP- ribosyltransferase (ADPRT) activity of the toxin. The region of DT encompassing His21 demonstrates sequence similarity with other toxins exhibiting ADPRT activity, is located along the catalytic cleft of DTA, and when His21 is chemically modified, ADPRT activity is abolished. His21 was mutagenized by a PCR-based system whereby all alternative amino acids were substituted in place of the histidine. The majority of the substitutions virtually abolished enzymatic activity, the exception being a mutant in which His21 was replaced with asparagine. this mutant demonstrated only a slight increase in Km and relatively small decreases in both reaction rate (kcat) and catalytic efficiency (kcat/Km). Asparagine is a sterically conserved substitution, but its side-chain is unable to replace the imidazole group of histidine in general acid-base mechanisms or to participate in electrostatic interactions. This suggests that His21 is important in maintaining a steric conformation required for catalysis rather than in participating in an electrostatic or acid-base type of exchange.
