Site-Directed Mutagenesis of Human Soluble Calcium-Activated Nucleotidase 1 (hSCAN-1):Identification of Residues Essential for Enzyme Activity and the Ca~(2+)-Induced Conformational Change

摘要

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects.This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases,and very little is known about the enzymatic mechanism.By multiple sequence alignment,eight regions that are highly conserved in the hSCAN-1 family were identified and named.To identify amino acids important for catalytic activity and enzyme specificity,seven point mutations were constructed,expressed in bacteria,refolded,purified,and characterized.Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities,while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity.Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity.Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities.Previously,our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity.Here we show that several mutants (D84A,R133A,and D151A) that lost most of their activity were unable to undergo the conformational change induced by Ca2+,as shown by Cibacron blue binding,limited proteolysis,and tryptophan fluorescence.We conclude that aspartic acid residues 84 and 151,as well as arginine residue 133,are essential for the Ca~(2+)-induced conformational change that is necessary for enzyme activity.Aspartic acid 175 and glutamic acid 130 are important for determining substrate specificity.In addition,we show that Sr~(2+),unlike Mg~(2+) and other divalent cations,can substitute for Ca~(2+) to induce the conformational change necessary for enzyme activity.However,Sr~(2+) cannot substitute for Ca~(2+) to support nucleotide hydrolysis,presumably because Sr~(2+) cannot substitute for Ca~(2+) in its second role as a nucleotide cosubstrate.The ramifications of our results on the interpretation of a recently published crystal structure are discussed.This information will facilitate future engineering of this enzyme designed to enhance its ability to hydrolyze ADP and thus increase its potential for therapeutic use in the treatment of pathological ischemic events triggered via activation of platelets by ADP.