Abstract
It has been proposed that the histidine at position 21 (H21) of the diphtheria toxin A subunit (DTA) plays an important role in the ADP-ribosyltransferase (ADPRT) activity of the toxin. The region of DT encompassing H21 demonstrates sequence similarity with other toxins exhibiting ADPRT activity, is located along the catalytic cleft of DTA, and when H21 is chemically modified, ADPRT activity is abolished. H21 was mutagenized by a polymerase chain reaction-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 H21 was replaced with asparagine (DTA-H21N). 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 H21 is important in maintaining a steric conformation required for catalysis rather than in participating in an electrostatic or acid-base type of exchange.
Highlights
It has been proposed that thehistidine at position21 (e.g. G52, G128) were identified using nitrosoguanidine muta
This suggests thatH 2 1 is impor- the potential role of H21 in the enzymatic activity of Diphtheria toxin (DT), we tant in maintaining a steric conformation required for initiated a series of studies aimed at the mutagenesis of this catalysis rather than in participating in an electrosrteastiidcue
Portion of the diphtheria toxinA subunit (DTA) gene was reaction; CRM, cross-reactingmaterial; PAGE, polyacrylamide gel elec- amplified using the second pair of primers (2b and 3) which made no trophoresis; NAD’, nicotinamide adenine dinucleotide; elongation factor 2 (EF-2), elonga- nucleotide substitutions, butgenerated a product whichhad 27 nucleotion factor 2; ADPRT,ADP-ribosyltransferase
Summary
Vol 269, No 6, Issue of February 11, pp. 4349-4354, 1994 Printed in U.S.A. (Received for publication, June 18, 1993, and in revised form, September 23, 1993). (H21) of the diphtheria toxinA subunit (DTA) plays an genesis (Uchida et al, 1973) Another postulated critical site important role in the ADP-ribosyltransferase (ADPRT) (E148) was identified by photoafinity labeling Portion of the DTA gene (nucleotides encoding residues 22-193) was reaction; CRM, cross-reactingmaterial; PAGE, polyacrylamide gel elec- amplified using the second pair of primers (2b and 3) which made no trophoresis; NAD’, nicotinamide adenine dinucleotide; EF-2, elonga- nucleotide substitutions, butgenerated a product whichhad 27 nucleotion factor 2; ADPRT,ADP-ribosyltransferase. ADP-ribosyltrunsferase Assay-~-[~~S]Methionine-labelepdroteins were quantitated by trichloroacetic acid precipitation and the enzymatic activities of the various DTA mutants were determined as previously described (Johnson et al, 1988).
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