380 2. ANALOGS OF ENZYME REACTION COMPONENTS 



D-altrose-6-P, glucuronate, and glucuronate-6-P. It may be noted that glu- 

 cose is the only hexokinase substrate that forms an inhibitory phosphate, 

 indicating the importance of the configuration at C-2 for inhibition. Thus 

 glucose phosphorylation in a closed system slows down progressively while 

 that of mannose is linear, a phenomenon which may be significant in 

 regulating the rate of sugar utilization. In a purer preparation of brain 

 hexokinase. Crane and Sols confirmed that the inhibition is not formally 

 competitive but that a reversible EI complex is formed. Phosphorylation 

 at C-6 (or at C-1 in the sorbose structure) seems necessary for inhibition; 

 e. g., glucose-1-P and 1,5-anhydro-D-glucitol lack inhibitory activity. It is 

 interesting that the inversion of the phosphate-carrying group at C-1 to 

 form /?-glucose-l,6-diP abolishes the inhibition, possibly due to a static 

 interference of the now closely apposed phosphate groups. Inversion of the 

 groups on C-2 (mannose-6-P), C-3 (allose-6-P), or C-4 (galactose-6-P) re- 

 duces or abolishes the inhibition; it was felt by Crane and Sols that the 

 configuration at C-3 influences the effect of an adjacent group and is not 

 directly concerned in the binding. It is difficult in most cases to decide if 

 the change in affinity on inversion of the groups is related to the hydroxyl 

 group as a binding site or as producing steric hindrance; thus the lack 

 of inhibition by galactose-6-P could be due either to the loss of hydrogen 

 bonding through the OH group (occurring in glucose-6-P) or to a protru- 

 sion of the OH group preventing approach of the pyranose ring. Compar- 

 ison with the corresponding deoxyglucose-6-P's might be informative. We 

 cannot do this for C-4, but at C-2 removal of the OH group (2-deoxy- 

 glucose-6-P) abolishes inhibition, pointing to the OH group as a binding 

 site. The weak inhibitory activity of 3-deoxyglucose-6-P substantiates the 

 idea that the 3-OH group is not involved in binding. The retention of 

 inhibition in l,5-anhydro-D-glucitol-6-P likewise indicates that the 1-OH is 

 not a binding site, but the loss of inhibition on C-1 methylation (a-meth- 

 ylglucoside-6-P) shows that steric repulsion occurs when the C-1 group 

 becomes too large. One may conclude that binding sites are at the 2-OH 

 and 6-phosphate groups, and possible at the 4-OH group. The inhibitors 

 thus attach to a different set of enzyme sites than the substrates, only 

 C-4 being common to both, and Crane and Sols visualized these differences 

 in the following structures, where the solid circles indicate necessary binding 

 positions: 



► H,OH •H2— O— PO",' 



H )r — \ 



OH 



Substrate Inhibitor 



