72 Martynas Ycas 



which certainly occur in proteins. These include hydroxylysine and hydro- 

 xyprohne (in collagen), phosphoserine (in a number of different proteins (15)), 

 thyroxine (in thyroglobulin) and tyrosine — O — sulphate (in fibrinogen) (16). 

 The distribution of these amino acids is different from the regular twenty. 

 Whereas the twenty amino acids occur in virtually all proteins, the super- 

 numerary ones have an erratic distribution, being confined to one or to a few. 

 The suggestion was first made by Crick, that the supernumerary amino acids 

 are the result of modifications of some of the regularly occurring amino acids 

 after these have been incorporated into a peptide chain. The biochemical 

 evidence for this is as follows. 



When one of the twenty regularly occurring amino acids is presented labeled 

 to an organism, it is rapidly incorporated into protein and most of the label 

 is found in the corresponding residue. It should be noted that glutamine and 

 glutamic acid are separately incorporated and do not arise one from another 

 by addition or subtraction of amide groups after incorporation (17). (A 

 similar demonstration for the analogous case of asparagine and aspartic acid 

 is still lacking.) Clearly, therefore, these amino acids are the precursors of 

 the corresponding protein-bound residues. 



The supernumerary amino acids behave differently. Thus lysine is the 

 precursor of hydroxylysine (18), but C^* or tritium-labeled hydroxylysine 

 is not incorporated into collagen (19). Similarly, proline is the precursor of 

 hydroxyprohne, but proline is a much better precursor of the hydroxyprolyl 

 of collagen than is hydroxyprohne itself (20, 21). These amino acids, then, 

 are not incorporated as such, but presumably are formed by oxidation of 

 protein-bound proline and lysine. Phosphoserine likewise is formed by phos- 

 phorylation of protein-bound serine (22). Thyroxine is apparently formed from 

 the tyrosine residues of thyroglobuhn (23). There is no information at present 

 on the metabolism of tyrosine — O — sulfate. 



Since not all appropriate residues are secondarily modified, this inter- 

 pretation imphes that the enzymes catalyzing such conversions show specificity 

 for sequence in the protein. At least one enzyme is known which shows such 

 specificity. Prostatic phosphatase dephosphorylates phosphoserine in the 

 sequence asx-serP-glx-ileu-ala, but not in glx-serP-ala (24). It is therefore 

 suggestive of some enzyme specificity that hydroxyprohne in collagen occurs 

 mainly, if not exclusively, before glycine (25) (Table IV). Other amino acids, 

 as shown later, shovv' no such neighbor preferences. The region determining 

 whether proline is to be oxidized or not probably includes more than three 

 residues, as indicated by the isolation from collagen of the tripeptides ala- 

 pro-gly; ala-Hpro-gly and ser-pro-gly; ser-Hpro-gly (Table IV). 



The biochemical evidence thus appears to indicate that the protein-forming 

 mechanism selects exactly twenty different kinds of amino acids, and that the 

 supernumerary ones arise by secondary modification of protein-bound residues. 

 A possible cause for error in this conclusion should be noted. It is virtually 

 certain that amino acids are not incorporated as such, but in the form of some 

 sort of activated derivative. If the same amino acid were to form more than 

 one derivative, the number of items to be selected would of course exceed 

 twenty. There is no evidence for this at present, and only further advances 

 in biochemistry can decide whether this is the case. 



