Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers.
There is considerable evidence that the mechanism of glucose transport by the transporter of human erythrocytes is one in which the transporter oscillates between two conformations, To and Ti. Each conformer possesses a single glucose binding site that in vivo faces either the extracellular space (conformer To) or the cytoplasm (conformer Ti). In this study, the interconversions of these conformers in the absence and presence of D-glucose have been directly observed by means of the stopped-flow method with fluorescence detection. Nearly unidirectional conversion of one conformer to the other was accomplished by rapidly mixing purified transporter (a mixture of To and Ti) with either 4,6-ethylidene-D-glucose, which preferentially binds to To, or phenyl beta-D-glucoside, which preferentially binds to Ti. The values of the individual rate constants for the conversion of Ti to To and vice versa in the absence and presence of D-glucose at 10.0 degrees C have been obtained, and these show that the kinetics are consistent with the alternating conformation model for transport. Conformational change occurs much more rapidly with glucose bound to the transporter. Furthermore, the activation energy Ea for conformer interconversion is much less when glucose is bound than for unliganded transporter. For example, Ea is approximately 28 kcal/mol for Ti----To versus 17 kcal/mol for Ti + S----ToS, where S is glucose. The alpha-anomer of glucose was 37% more effective than the beta-anomer in speeding the interconversion.(ABSTRACT TRUNCATED AT 250 WORDS)
Appleman
J R
JR
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Lienhard
G E
GE
D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature
D009004:Monosaccharide Transport Proteins; C013984:ethylidene glucose; D005947:Glucose