Direct evidence of conformational heterogeneity in human pancreatic glucokinase from high-resolution nuclear magnetic resonance.
High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.
Larion
Mioara
M
Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306-4390, USA.
Salinas
Roberto K
RK
Bruschweiler-Li
Lei
L
Brüschweiler
Rafael
R
Miller
Brian G
BG
D005941:Glucokinase / Q000737:chemistry* / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry; D006801:Humans; D008279:Magnetic Resonance Imaging; D008968:Molecular Conformation; D010179:Pancreas / Q000378:metabolism
D005941:Glucokinase; D005947:Glucose