408 2. ANALOGS OF ENZYME REACTION COMPONENTS 



because the inhibiting form could represent only a small fraction of the total 

 concentration of the inhibitor. 



Glyceraldehyde-3-Phosphate Dehydrogenase 



A new, potent, and apparently specific inhibitor of this enzyme has 

 been found and is likely to be useful as a blocking agent of this step in 

 glycolysis. Glycolaldehyde-2-P freshly prepared does not inhibit glyceral- 

 dehyde-3-P dehydrogenase, but either aging the preparation or allowing it 

 to react in 1 N NaOH for a short time leads to the formation of a potent 

 inhibitor, termed tetrose-diP by Racker et al., (1959) and isolated as d- 

 threose-2,4-diP by Fluharty and Ballou (1959). Both the d- and L-isomers 



CHO 



of the inhibitor were synthesized by the latter workers and only the D-iso- 

 mer was found to inhibit strongly. The inhibition is reversible but non- 

 competitive with respect to glyceraldehyde-3-P; the inhibition may actually 

 increase with NAD concentration. The value of K, for rabbit muscle en- 

 zyme is 0.0001-0.0002 milf. D-Threose-2,4-diP is oxidized by the enzyme 

 but only as much as the NAD present. Fluharty and Ballou postulated 

 that it might react with a site other than the normal catalytic site for 

 oxidation of glyceraldehyde-3-P but Racker etal., showed spectroscopically 

 that a stable acyl-enzyme complex is formed, probably with the SH group 

 known to be involved in the catalysis and the binding of NAD. The sub- 

 strate reactions might be written as: 



Enzyme-NAD -f Glyceraldehyde-3-P :f± phosphoglyceryl-enzyme-NADH 

 Phosphoglyceryl-enzyme-NADH + P 5± glycerate-l,3-diP + enzyme-NADH 



whereas the reaction with the inhibitor is: 



Enzyme-NAD + threose-2,4-diP :^ diphosphothreonyl-enzyme-NADH 



Normally an inorganic phosphate is transferred from a second site to the 

 phosphoglyceryl-enzyme to form glycerate-l,3-diP, but the location of the 

 2-phosphate group on the inhibitor is such as to occupy this second site 

 so that no phosphate can enter the reaction. This is thus an example of 

 an inhibition in which a substance enters the reaction sequence in the same 



