Role of connecting loop I in catalysis and allosteric regulation of human glucokinase.
Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady-state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47-71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C-terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the kcat value unchanged; however, removing three or more residues reduces kcat by 1000-fold. In contrast, the glucose K0.5 and KD values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline-66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK-I) eliminates cooperativity without impacting the kcat and glucose K0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.
Department of Chemistry and Biochemistry, 4005 Chemical Sciences Laboratory, Florida State University, Tallahassee, Florida, 32306-4390.
Juliana A
JA
Martinez
Mioara
M
Larion
Maria S
MS
Conejo
Carol M
CM
Porter
Brian G
BG
Miller
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D002384:Catalysis; D020134:Catalytic Domain; D005941:Glucokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
C472074:HK1 protein, human; D006593:Hexokinase; D005941:Glucokinase
allostery; glucokinase; hysteresis; kinetic cooperativity