Direct and indirect roles of His-418 in metal binding and in the activity of beta-galactosidase (E. coli).
The active site of ss-galactosidase (E. coli) contains a Mg(2+) ion ligated by Glu-416, His-418 and Glu-461 plus three water molecules. A Na(+) ion binds nearby. To better understand the role of the active site Mg(2+) and its ligands, His-418 was substituted with Asn, Glu and Phe. The Asn-418 and Glu-418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu-418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg(2+) site also reduce Na(+) binding affinity. The Phe variant had reduced stability, bound Mg(2+) weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His-418, together with the Mg(2+), modulate the central role of Glu-461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His-418 is very important for the formation of allolactose (the natural inducer of the lac operon).
Juers
Douglas H
DH
Instititute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene, OR 97403-1229, USA.
Rob
Beatrice
B
Dugdale
Megan L
ML
Rahimzadeh
Nastaron
N
Giang
Clarence
C
Lee
Michelle
M
Matthews
Brian W
BW
Huber
Reuben E
RE
D019943:Amino Acid Substitution; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005947:Glucose / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D006639:Histidine; D012964:Sodium; D001616:beta-Galactosidase; D008274:Magnesium; D005947:Glucose