STRUCTURE AND FUNCTION OF SOME ENZYMES 



143 



s 



o 



.3 ^ 



a; 



i^ J 



O / 



5 -S;;!? £70 



310 350 390 m WO SlOmji 



Fig. lb. Absorption spectrum of the lumiflavin 



(O — O) and of the flavin-adenine-dinucleotide 



(•— •) (Warburg and Christian). 



Wave length in m|j, 



flavin is decreased by the linking to the pro- 

 tein, or, in other words, the prosthetic group 

 is activated by the specific protein. 



If now salt-free solutions of the ferment 

 were dialyzed at 0° against weak hydro- 

 chloric acid the ferment was split up into 

 the prosthetic group, which passed through 

 the membrane, and a colorless protein, 

 which remained within the cellophane tube. 

 The protein was found to be denatured 

 after this procedure, but could to a great 

 extent be made native again by dialysis 

 against water. If this protein were mixed 

 with the prosthetic group, the yellow fer- 

 ment appeared again (see Fig. 2). 



_-> 

 70 '-'^ 



o 



60 

 50 



to 



30 

 20 

 10 



t¥ 2,8 ¥,2 5,6 7,0 8,t 9,8 11,2 12,6 1¥,0 15,¥ 



Fig. 2. Resynthesis of the yellow ferment. The 



catalytic activity on adding increasing amounts of 



the prosthetic group to a constant amount of the 



protein component. 



Determinations of the catalytic activity 

 in the test system (oxygen ; yellow ferment ; 

 triphosphopyridine proteid ; Robison ester), 

 as well as of the isoelectric point and of 



the sedimentation constant, gave the same 

 values as with the ferment that had never 

 been split. So it was proved that the yel- 

 low ferment had been submitted to a really 

 reversible cleavage. It was the first time 

 that an enzj^me had been split into a pros- 

 thetic group and a protein, and resynthe- 

 sized again. The experiment constituted 

 a direct proof of the correctness of the old 

 theory that enzymes consist of two radi- 

 cally different parts, a so-called active 

 group and a bearer. The chemical consti- 

 tution of the ' ' old ' ' yellow ferment has been 

 clarified as far as possible — and that means 

 to 0.65 per cent, which is the percentage of 

 the prosthetic group. The composition of 

 the protein part, 99.35 per cent of the 

 molecule, is of course as unknown as that 

 of all other proteins. 



The clearing up of the composition of 

 lactoflavin and lactoflavin phosphoric acid 

 was carried out, after the fundamental 

 work of Warburg and Christian, mainly by 

 Karrer and his co-workers in Zurich and by 

 Kuhn and his co-workers in Heidelberg. 

 Their investigations led to the formula 

 presented on page 144. 



It is a most interesting fact that each of 

 the many parts of the molecule of the pros- 

 thetic group exerts its own influence upon 

 the properties of the enzyme. 



The nitrogen atoms (1) and (10) are the 

 carriers of the hydrogen stream from the 

 substrate according to the formula : 



By means of the "one-step "-reaction of 

 Michaelis the reduction of the yellow form 

 proceeds with one hydrogen atom at a time 

 over the monohydroradical to the leuco 

 form, or vice versa. The transference of 

 one hydrogen atom at a time must be a 

 more probable, and thus faster, reaction 

 than the direct reduction to the leuco form 

 by means of two hydrogen atoms simul- 

 taneously. The monohj'droradical of the 

 free riboflavin can exist only in a strongly 

 acid solution. The yellow ferment, on the 

 contrary, seems to give a monohydroradical 

 that can exist in neutral solution (Haas). 

 Perhaps this is the mechanism of "activa- 

 tion" of the lactoflavin phosphoric acid 

 by the protein component. It seems most 



