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Alanine mutations of the proposed catalytically essential residues in his-toaspartic 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)Proteins55, 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)Biochemistry43, 14521–14528] | ễFEBS Journal The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease Charity L. Parr1 Takuji Tanaka2 Huogen Xiao1 and Rickey Y. Yada1 1 Department of Food Science University of Guelph Canada 2 Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon Canada Keywords active site histoaspartic protease malaria model plasmepsin Correspondence R. Y. Yada Department of Food Science University of Guelph Guelph Ontario Canada N1G 2W1 Fax 1 519 824 6631 Tel 1 519 842 4120 ext 58915 E-mail ryada@uoguelph.ca Received 16 October 2007 revised 9 January 2008 accepted 7 February 2008 doi 10.1111 j.1742-4658.2008.06325.x Alanine mutations of the proposed catalytically essential residues in his-toaspartic 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 wildtype HAP H34A and S37A were strongly inhibited by the serine .
