Structure-based design of a lysozyme with altered catalytic activity.
Here we show that the substitution Thr 26-->His in the active site of T4 lysozyme causes the product to change from the alpha- to the beta-anomer. This implies an alteration in the catalytic mechanism of the enzyme. From the change in product, together with inspection of relevant crystal structures, it is inferred that wild-type T4 lysozyme is an anomer-inverting enzyme with a single displacement mechanism in which water attacks from the alpha-side of the substrate. In contrast, the mutant T26H is an anomer-retaining enzyme with an apparently double displacement mechanism in which a water molecule attacks from the opposite side of the substrate. The results also show that the mechanism of wild-type T4 lysozyme differs from that of hen egg-white lysozyme even though both enzymes are presumed to have evolved from a common precursor.
Kuroki
R
R
Institute of Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene 97403, USA.
Weaver
L H
LH
Matthews
B W
BW
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005075:Biological Evolution; D002240:Carbohydrate Sequence; D002384:Catalysis; D003461:Crystallography; D004529:Egg White; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015202:Protein Engineering; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D006026:Glycoside Hydrolases; D009113:Muramidase