The Chemistry of Proteolytic Enzymes

The study of the relationship between the genetic evolution, the structure and the function of enzymes is given a variety of names, such as molecular biology, molecular genetics, and evolutional biochemistry. In our Laboratory we started from organic chemistry, and we try to use the methods of chemistry as much as possible in this field of biologically active macromolecules. About ten years ago, the main task of the chemist working with an enzyme was to determine the amino acid sequence of its polypeptide chain and to identify, by means of chemical modifications, the groups responsible for the biological activity. Then came the most valuable intervention of x-ray crystallographers who were able to trace the spatial arrangement of hundreds of amino acids forming the whole macromolecule. The answer to the question bow the actual conformation of the macromolecule influences the activity of an enzyme has now become urgent. Today the chemist is no longer faced with the problem of following merely a reaction mechanism involving one or two functional groups, but has to consider a concerted effect of many groups in environments of hydrophobic and hydrophilic interactions. Such groupings fixed in a very rigid conformation form clefts and holes on the surface of a macromolecule. Recently an eminent biochemist said that we have to deal nowadays with a special branch of chemistry, the chemistry of holes. A part of this review can thus be considered as a contribution to this field. It is about twenty years ago that Sanger's pioneer work established the complete amino acid sequence of insulin. This new information brought about the collapse of many old theories on the structure of proteins. The sequences of amino acids in the two chains of insulin showed no regularities or clear-cut pattern. Moreover, it was impossible to explain the hormonal activity from the primary structure. It became clear that if there existed any regularities or patterns in the structure of proteins, they would hardly be detectable without knowledge of the exact chemical architecture of many proteins. There were two lines of chemical approach to the question whether there exist structural similarities between related proteins or not: to study the amino acid sequences either of functionally similar proteins from different organisms, or of functionally different proteins from the same cell. Cytochrome C and later the haemoglobins from different organisms have attracted much interest. Studies on these substances have revealed the rules governing the differentiation of protein structures in the course of biological evolution of species. Fourteen years ago, in our Laboratory in Prague, we started to follow the second line of approach, that is, we looked for related proteins formed in the same cell. These efforts were aimed at experimental verification of the