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Alkaline phosphatase from psychrophile TAB5 and cold‐adapted, northern shrimp (Pandalus borealis) are structurally similar yet functionally distinct
Author(s) -
Post Marc Alan
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a479-d
Subject(s) - tap water , psychrophile , tris , enzyme kinetics , chemistry , alkaline phosphatase , ic50 , shrimp , phosphate , biochemistry , biology , enzyme , in vitro , active site , ecology , environmental engineering , engineering
Kinetics and enzymology of alkaline phosphatase (AP) from cold water adapted Northern shrimp ( Pandalus borealis ) and from the psychrophilic Antarctic bacterium TAB5 were compared in order to correlate functional nuances with published structures. Activity was determined ising p ‐nitrophenyl phosphate. TAB5 AP (TAP) activity required both Zn 2+ and Mg 2+ ; shrimp AP (SAP) activity required Zn 2+ but not Mg 2+ , however activity was enhanced by Mg 2+ . SAP activity was inhibited by elevated Zn 2+ (IC 50 =11mM) but TAP activity was not. TAP activity was inhibited by, but SAP activity was enhanced by, NaCl or KCl. TAP was inhibited by SDS (IC 50 =0.015%); while SAP was not inhibited by detergents. The pH opt of SAP was 10.5 in the absence of Tris‐HCl, but in its presence the pH opt was 8. The pH opt of TAP was 9 in the absence of Tris‐HCl, but in its presence the pH opt exceeded 11. K M values for SAP and TAP were 690μ M and 530μ M, respectively. V MAX values for SAP and TAP were 3.9μ mol·min− 1 ·μ g− 1 and 0.059μmol·min μ − 1 ·μ g− 1 , respectively. The k cat /K M ratio for SAP was 100‐fold greater than for TAP (1.04·10 7 sec− 1 ·M− 1 vs 1.51·10 5 sec− 1 ·M− 1 , respectively). Rate of thermal denaturation was similar at 65°C (t ½ =8.6s for SAP vs 4.2s for TAP) but differed at 37°C (t ½ =90min for SAP vs 28min for TAP). In E. coli , an association between K328 and V MAX and Tris interactions has been reported. This residue is histidine in SAP and tryptophan in TAP, the different residue identities correlating with the different functional observations. Functional effects of salts, reducing agents, and detergents were noted for their implications for the use of SAP and TAP. Support from USB Corporation.