The importance of histidine residues in human ecto-nucleoside triphosphate diphosphohydrolase-3 as determined by site-directed mutagenesis.
Most ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are inhibited by the histidine reagent diethyl pyrocarbonate (DEPC), while being resistant to inhibition by many other chemical modification agents. We used site-directed mutagenesis to investigate the sites of modification responsible for DEPC inhibition. First, we constructed the mutations H135A and R67H in eNTPDase-3 to address the possibility that, in eNTPDase-3, histidine 135 compensates for the lack of a histidine in apyrase conserved region (ACR) 1, present in all other membranous eNTPDases (but replaced by R67 in ACR1 of eNTPDase-3). We found histidine 135 is a major, but not the sole, target for DEPC-induced inhibition in eNTPDase-3. In addition, analysis of the R67H mutant led us to conclude that this site is important for DEPC inactivation of other eNTPDases. We also mutated singly and collectively three of the most conserved histidine residues present in eNTPDase-3 (129, 257 and 447) to alanine. None of the single, conserved histidine mutations nor the triple histidine mutation inactivated the enzyme or decreased susceptibility to DEPC inhibition. However, changes in the tendency of monomers to self-associate were noted, and the triple histidine mutant exhibited a higher nucleotidase specific activity than the wild-type.
Hicks-Berger
C A
CA
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, OH 45267-0575, USA.
Yang
F
F
Smith
T M
TM
Kirley
T L
TL
D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000737:chemistry; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*
D015703:Antigens, CD; D006639:Histidine; D011755:Pyrophosphatases; C092064:ectonucleotide pyrophosphohydrolase; D001081:Apyrase; C067869:CD39 antigen; D004047:Diethyl Pyrocarbonate