106 - The Cell 



to demonstrate the formation of a catalase- 

 H^O;. intermediary compound, even though 

 the life of this unstable complex endures for 

 only 1/85,000 of a second. Britton Chance, 

 of the University of Pennsylvania, also found 

 that peroxidase, a brown enzyme extracted 

 from horse-radish, forms two successive inter- 

 mediary complexes, one green and the other 

 pale red, when FF,G\ is added to the system; 

 and subsequently a number of other enzyme- 

 substrate intermediaries have been revealed. 



Concept of Intermolecular Fit. The "lock 

 and key concept" of enzyme-substrate fit was 

 first proposed by the great German biochem- 

 ist, Emil Fischer, almost 75 years ago. A de- 

 tailed knowledge of the molecular structure 

 of proteins has been difficult to gain, how- 

 ever, and evidence supporting this hypothesis, 

 though very suggestive and intriguing, is not 

 generally very specific. However, there are 

 many cases where such an interpretation 

 seems applicable. F. S. G. Barron at Chicago 

 University, for example, studied the enzyme 

 system that prepares acetic acid (CH.COOH) 

 for oxidation by the Krebs cycle enzymes 

 (Fig. 8-5). Barron found that very small modi- 

 fications in the configuration of the substrate 

 molecule had a decisive influence in determin- 

 ing whether it could be handled by the en- 

 zyme. If, instead of acetic acid (CH :i COOH), 

 fluoroacetic acid (F-CHXOOH) was pre- 

 sented to the enzyme, a stable enzyme- 

 substrate compound was formed, which 

 blocked the catalytic focus of the enzyme. 

 On the other hand, chloroacetic acid 

 (CUCHXOOH) had little or no effect. 

 Barron concluded that probably the length 

 of the interatomic bonds, namely carbon to 

 hydrogen (1.09 Angstroms), carbon to fluo- 

 rine (1.41 Angstroms), and carbon to chlorine 

 1.76 Angstroms), played a decisive role in 

 determining the fit — between the enzyme 

 and substrate and between the enzyme and 

 the inhibitor. 



Many other examples can be given to illus- 

 trate the concept of fit, semifit, and misfit 

 between specific enzymes, substrates, and in- 

 hibitors. The protein-hydrolyzing enzyme, 



aminopeptidase (Table 5-1), fits only amino 

 acids having an exposed amino (NFL) group, 

 and these must be situated at the end of a 

 peptide chain; another pancreatic enzyme, 

 carboxy peptidase, (Table 5-1), can only split 

 off amino acids having an exposed carboxyl 

 (— COOH) group, and these likewise must be 

 terminal in the chain; trypsin (Table 5-1) 

 splits protein chains only at certain spe- 

 cific linkage points, whereas chymotrypsin 

 achieves rupture at other different points; 

 and in each case the site of rupture appears 

 to be determined by the configuration of the 

 specific amino acids that adjoin the linkage. 

 Moreover, cases of semifit, where a slightly 

 modified substrate affixes itself firmly to the 

 catalytic area of an enzyme and blocks oft 

 further catalytic activity (Fig. 5-3), have been 

 studied in various ways. An outstanding ex- 

 ample, in fact, is provided by the very useful 

 drug, sulfanilamide. The molecular configu- 

 rations of this compound and of an essential 

 (vitamin) substance present in many cells 

 (pfl)Y(-aminobenzoic acid) are very similar 

 (Fig. 5-4). Thus it is not surprising to find 

 that sulfanilamide kills bacteria by blocking 

 oft the enzyme system that normally would 

 handle the synthesis of p</)Y<-aminobenzoic 

 acid in the bacterial cell. 



ENZYME 



E-l COMPLEX 



E + ! 



E-l 

 STABLE 



Fig. 5-3. Enzyme inhibition: concept of molecular fit. 

 The inhibitor (I) unites very firmly with the enzyme, 

 blocking off its catalytic site. When such union is rela- 

 tively weak, and when the concentration of inhibitor 

 is low, the inhibitor competes with the substrate for 

 occupation of the catalytic site. This results in partial, 

 or competitive, inhibition. 



