HENRY R. MAHLER 



This is, of course, the approach used in the second of our 

 methods: the isolation, purification, and exhaustive study of 

 the individual enzymes involved in a mitochondrial reaction 

 sequence. In general the next step is an attempt at recon- 

 structing the sequence by the use of a combination of the 

 individual enzymes, their coenzymes, cofactors, etc. These 

 steps were used with spectacular success in the elaboration 

 and reconstruction of the three great metabolic cycles: the 

 tricarboxylic acid cycle (32), the urea cycle (35), and the fatty 

 acid cycle (15,23), as well as such other complex processes as 

 purine synthesis. This is the method par excellence for arriving 

 at a complete appreciation of each individual enzymatic activity 

 and of its potentialities. Note first that I say enzymatic activity 

 and not enzyme. The two are not necessarily synonymous. An 

 enzymatic activity is determined by the assay system employed 

 in its measurement. It is of course associated with a definite 

 protein component — but whether this component as such had 

 an equally definite existence prior to its isolation, or whether it 

 originally formed part of a diflferent or of a larger molecular or 

 catalytic unit may not always be readily discernible or even 

 capable of determination. This point of view will become less 

 obscure as the discussion proceeds, but it must be apparent that 

 the use of artificial electron acceptors in oxidative reactions 

 (such as dyes and quinones), a usual, nay a necessary, experi- 

 mental device, does not lead to the isolation of "natural" 

 enzymes or at least does not need to. For the introduction of an 

 "artificial" acceptor imposes an element of artificiality into the 

 system, and as far as the study of an oxidative enzyme is con- 

 cerned, an artificial acceptor is no whit different from the use 

 of an artificial substrate or an artificial coenzyme. Yet fre- 

 quently we do not even know the nature of the "natural" 

 acceptor, and thus we find ourselves impaled upon the horns of 

 a dilemma from the very outset. 



The second point to be raised here concerns the use of the 

 word potentialities. The study of the mechanism and the fine 

 structure of individual enzymatic activities is limited only by our 



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