INTERACTION FORCES IN VARIOUS ENZYME SYSTEMS 295 



constants are not accurately known, and furthermore they depend strongly 

 on the concentrations of ions, particularly Ca"*""^ and Mg"^"*". Blum (1955) 

 determined the value of ^atp for rabbit myosin ATPase in the presence of 

 1 mM Ca"^"^ to be 6.25 XlO~^ mM; from inhibition experiments the Kadp 

 under the same conditions was calculated to be 1.64 Xl0~^ n\M. This cor- 

 responds to a difference in binding energy of 1.93 kcal/mole. Since AMP 

 inhibits much more weakly than ADP, its binding energy must be lower 

 than for ADP and probably of a similar order of magnitude to the ATP- 

 ADP difference. This would imply that the two terminal phosphate groups 

 participate in the binding; nothing can be concluded about the proximal 

 phosphate group. 



Interaction Energies of Sarcosine Oxidase Inhibitors 



Sarcosine is oxidized to formaldehyde and glycine in a suspension of 

 liver mitochondria; this reaction is competitively inhibited by methoxyace- 

 tate, the oxygen analog of sarcosine, and many related compounds (Frisell 

 and Mackenzie, 1955). In Table 6-27 is shown the inhibition produced 

 by 20.8 mM inhibitor when sarcosine is 8.33 mM. The relative binding 

 energy is calculated from the inhibition constants obtained from Eq. 3-13: 



(I) (1 - 



^'- = n iU^/z.-^ • (<5-118) 



[1 + (b)/A,] I 



Since the pH was 7.8, the carboxyl groups are ionized and it is likely that 

 the amino groups and — "^NHo— group of sarcosine are protonated. The 

 active site of sarcosine oxidase is pictured in Fig. 6-2; a three-point attach- 

 ment of sarcosine was suggested by Frisell and Mackenzie, the carboxylate 

 group interacting with a cationic protein group, the terminal methyl 

 group being attracted to a protein hydrocarbon residue by dispersion for- 

 ces, and the — "^NHg— group reacting with some electrophilic radical on 

 the protein. 



Comparing acetate and ethanol, methoxyacetate and methoxyethanol, 

 and sarcosine with sarcosine amide, the ion-ion carboxylate binding energy 

 would be about 1.8 kcal/mole, implying an equilibrium distance of 5.3 A, 

 similar to values obtained on other enzymes for such interactions. Com- 

 parison of sarcosine with butyrate (assuming that tlie K„j for sarcosine 

 represents the true dissociation constant) indicates that the replacement 

 of a — CH.,— group by a — "^NHa— group leads to 1.59 kcal/mole greater 

 binding energy; the groups —0— and — S— also increase the binding but 

 not so much. It would appear likely that hydrogen bonding is responsible; 

 this would argue for the nitrogen group in sarcosine being uncharged as 

 — NH— , which would be substantiated by the small binding of glycine, 

 but the i:)K^ for this group is given as 10.01 (Cohn and Edsall, 1943, p. 85) 



