Premium
The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease
Author(s) -
Parr Charity L.,
Tanaka Takuji,
Xiao Huogen,
Yada Rickey Y.
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06325.x
Subject(s) - plasmodium falciparum , protease , active site , virology , chemistry , catalysis , malaria , biology , biochemistry , enzyme , immunology
Alanine mutations of the proposed catalytically essential residues in histoaspartic protease (HAP) (H34A, S37A and D214A) were generated to investigate whether: (a) HAP is a serine protease with a catalytic triad of His34, Ser37 and Asp214 [Andreeva N, Bogdanovich P, Kashparov I, Popov M & Stengach M (2004) Proteins 55 , 705–710]; or (b) HAP is a novel protease with Asp214 acting as both the acid and the base during substrate catalysis with His34 providing critical stabilization [Bjelic S & Aqvist J (2004) Biochemistry 43 , 14521–14528]. Our results indicated that recombinant wild‐type HAP, S37A and H34A were capable of autoactivation, whereas D214A was not. The inability of D214A to autoactivate highlighted the importance of Asp214 for catalysis. H34A and S37A mutants hydrolyzed synthetic substrate indicating that neither His34 nor Ser37 was essential for substrate catalysis. Both mutants did, however, have reduced catalytic efficiency ( P ≤ 0.05) compared with wild‐type HAP, which was attributed to the stabilizing role of His34 and Ser37 during catalysis. The mature forms of wild‐type HAP, H34A and S37A all exhibited high activity over a broad pH range of 5.0–8.5 with maximum activity occurring between pH 7.5 and 8.0. Inhibition studies indicated that wild‐type HAP, H34A and S37A were strongly inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride, but only weakly inhibited by pepstatin A. The data, in concert with molecular modeling, suggest a novel mode of catalysis with a single aspartic acid residue performing both the acid and base roles.