Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering.
On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed in Escherichia coli, was purified and characterized. The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group. The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme. Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2. When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2. However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group. Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering.
Bhat
M K
MK
Department of Protein Engineering, AFRC Institute of Food Research, Reading Laboratory, U.K.
Pickersgill
R W
RW
Perry
B N
BN
Brown
R A
RA
Jones
S T
ST
Mueller-Harvey
I
I
Sumner
I G
IG
Goodenough
P W
PW
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004530:Egg Yolk; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007930:Leucine / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013552:Swine
D017931:DNA Primers; D005971:Glutamates; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D007930:Leucine