80 



The Nature of Biological Diversity 



actions involve function of discrete, covalently bound intermediates 

 in their catalyses. Such is not the case with the kinases. Of the various 

 experimental results pointing to the absence of a phosphoryl enzyme 

 intermediate in the kinase reactions, results of one recent approach 

 (28) may be of interest because they allow definitive conclusions 

 and because they illustrate a simplicity of experimental design based 

 on the use of comparatively large amounts of a given enzyme. The 

 findings of such an experiment with pyruvate kinase are given in 



0.62 



0.58 



o 

 to 



0.54 



0.50 



~l I I | I I I 1 J 



^Lactate Dehydrogenase plus 

 3.2 xlO -7 M Pyruvate Kinase 



i — i — r 



(MIXING) 

 Pyruvate Kinase 



3.2 x I0" 6 M 



3.3 x I0~ 6 M 



J I L 



_l__l L 



AA = 0.021 



J I L 



J I L 



5 10 15 



Minutes 



FIG. 3. Demonstration of lack of phosphoryl enzyme formation by pyruvate kinase. 



Fig. 3. The system used is actually a sensitive test for formation of 

 ADP. The initial reaction mixture contains phosphoenolypyruvate, 

 ATP, and reduced diphosphopyridine nucleotide, together with small 

 traces of ADP and pyruvate. Upon addition of catalytic amounts of 

 pyruvate kinase and lactate dehydrogenase, practically all the ADP 

 present is phosphorylated to give ATP through the pyruvate kinase 

 reaction, with maintenance of a high ATP/ADP ratio. The pyruvate 

 formed from phosphoenolpyruvate oxidizes some DPNH, with a 

 resultant slight decrease in absorbancy at 340 millimicrons. The 

 absorbancy as measured on a sensitive recording spectrophotometer 



