THE NATURE OF ENZYME ACTIVE CENTERS 195 



or underlying the active center, although not directly concerned in the 

 reaction, can influence the activity of the center through steric or elec- 

 trostatic effects. The total protein, nevertheless, is apparently not neces- 

 sary for activity because active fragments have been obtained from several 

 proteins (aldolase, papain, ribonuclease, trypsin, and pepsin) and the intact 

 structure of the total protein is at least sometimes of little significance 

 since cross-linkages between polypeptide chains have been broken without 

 loss of activity (e.g., pepsin S-S bridges), The behavior of ribonuclease is 

 particularly interesting inasmuch as pepsin inactivates it rapidly by re- 

 moval of a tetrapeptide asp-ala-ser-val whereas carboxypeptidase removes 

 the tripeptide ala-ser-val without inactivation, from which it is apparent 

 that the aspartate residue is involved in the active site which is near the 

 C-terminal end of the polypeptide chain (Anfinsen, 1956). The most ex- 

 tensive degradation of an enzyme without loss of activity is the removal of 

 two-thirds of the 180 amino acid residues from papain by leucine amino- 

 peptidase (Hill and Smith, 1956). 



Most enzymes investigated by end-group analysis contain only one poly- 

 peptide chain although some undoubtedly possess more. The configuration 

 of the chain in enzymes, particularly at the active center, is generally 

 unknown. Parts of the polypeptide chain are probably hydrogen-bonded 

 into helices, but not all because both X-ray and optical dispersion data in 

 the few cases studied have indicated that only a fraction of the protein is 

 helical; in ribonuclease only 15% of the protein is helical (Yang and Doty, 

 1957). The nonhelical portions are not randomly oriented but are undoubt- 

 edly in specific configurations determined to a great extent by the looping 

 of the polypeptide chains through cross-linkages, such as the disulfide 

 R-S-S-R' (ribonuclease has four), phosphate R-O-P-OR' (pepsin has one), 

 or pyrophosphate R-0-P-O-P-O-R'. Hydrogen bonding can also occur in 

 nonhelical structures and the nonpolar cohesive forces between hydro- 

 carbon side-chains are often stronger than generally realized. 



The presence of certain amino acids at an active site can be demonstrated 

 or inferred by a variety of techniques: the reaction of amino acid side-chains 

 with group-specific reagents, a reduction or loss of activity indicating that 

 the particular group is associated with the active site, but not necessarily 

 proving it; the isolation of amino acid or peptide fragments containing a 

 stable inhibitor; the subjection of the enzyme to proteolysis; and the de- 

 termination of the ionization constants of active site groups from the va- 

 riation of enzymic activity with pH. Sulfhydryl groups are recognized as 

 present in many active sites because of the marked inhibition by sub- 

 stances reacting quite specifically with such groups. The use of fluorophos- 

 phonates, such as DFP, has led to the isolation of phosphoserine peptides 

 from trypsin and chymotrypsin; the amino acid sequence for short distances 

 on either side of serine can be determined, although the relationship of 



