Friday, 17 October 2014

Having spoken this week to students about the Science, the Scientists and the Significance of Nobel Prizes from the first to the last, the work of Pauling and Sanger came to the fore. I thought I would therefore provide a link from one of the earliest Nobel prizes (here I am referring to Emil Fischer's ground-breaking work on peptides, even though his award was for his work on sugars and purines! [NP 1902]) through Linus Pauling's work on the chemical bond, including the peptide bond [NP 1954, ], Fred Sanger's first Nobel Prize [NP 1958] for his work on the structure of proteins, and finally to the NP 2009 awarded to Yonath, Steitz and Ramakrishnan for determining the structure of the macromolecular machine that is responsible for peptide bond synthesis in all living organisms: the Ribosome. These prizes provide a great opportunity to consider, Biology, Chemistry and Physics from an experimental and theoretical standpoint. They also illustrate the enduring influence of work of Nobel Laureates. I shudder to think of the intellect of Emil Fischer (top left),who carried out his work in such "low-tech" circumstances, unencumbered by over 100 years of massive research outputs in Science!

One of the early concepts that students of Biochemistry have to learn is that the amino acids that make up proteins are only of the L form: the D form is found in Nature, but the L form has emerged after many years of evolution as the "enantiomer" found in proteins. You can read the basics on amino acids at this wikipedia site. Understanding the chemical linkage between amino acids occupied the mind of Fischer over ten years at the turn of the last century. Having obtained several amino acids in an optically pure form, he synthesised several dipeptides and laid the foundation for our understanding of the peptide bond. There is a nice little Blog post on the peptide bond here, by "Sandwalk" at the University of Toronto. The figure above (RHS) illustrates the bonds in a polypeptide chain and in particular the contrast between the rotational freedom at bonds adjacent to the peptide bond. It was largely through work by Linus Pauling (and Robert Corey) that the rigidity and partial double bond character of the peptide bond was understood. There is a nice historical sketch to be found at Edison, where he discusses the resonance stabilisation that underpins the nature of the peptide bond. In essence, the distribution of electrons of the peptide bond, is so energetically favourable that it essentially fixes the polypeptide chain and constrains the secondary structure of a protein. This, along with the rule that hydrogen bonds between amino and carbonyl pairs prevail, provided Pauling and colleagues with the impetus to predict the formation of alpha helices and beta sheets in protein structures. Pauling was proved right some years later by X ray crystallography from the Cambridge Nobel laureates,  Max Perutz and John Kendrew.

It is worth taking a moment to reflect on the intellect of Pauling, notwithstanding his contributions to enzyme catalysis and other aspects of chemical bonding. The coalescence of his knowledge of chemical bonds, both covalent and hydrogen, together with an ability to identify patterns and deliver a robust visual expression through molecular modelling, is in my view quite formidable! If you are interested in Pauling, you might look here and an interesting intellectual exercise is to read the original papers from Pauling and Watson and Crick, side by side, on the interpretation of experimental data that led to the proposals for the structure of DNA. We take the structure of DNA for granted, but working with so little data, these two interpretations demonstrate how challenging it is when you are tackling a problem of immense importance.

To summarise, the work of Fischer (and of course others) in establishing the chemical linkage between amino acids followed three decades later by Pauling's insights regarding the conformational characteristics of the peptide bond, provided Biochemists and X ray crystallographers with a framework for elucidating the three dimensional properties of Proteins (the image on the RHS is John Kendrew's original model of Myoglobin: the "sausages" are Pauling's alpha helices). Let us not forget here the importance of Fred Sanger's contribution in proving by his elegant chemistry, the uniqueness of the primary structure of proteins! Now  let us look at two further aspects of the life of the peptide bond: its formation via the ribosome and its hydrolysis through the action of proteases. I shall confine myself to the Serine Proteases in this Blog. 

The active site of Trypsin
The energy locked up in the peptide bond is approximately 3kcal/mol, although there are known to be differences arising say when one amino acid is added to another compared with consecutive additions. Moreover there will be local variations in the susceptibility of some peptide bonds to hydrolysis caused by factors such as local structure and side chain chemistry. [When thinking about biosynthesis, the hydrolysis of ATP and GTP generates significantly more energy, but not all is channelled into bond formation, some is used to drive the movement of the ribosome along the mRNA (among other essential reaction steps)]. The Serine proteases include the enzymes Trypsin, Chymotrypsin and Elastase: "The Holy Trinity" of undergraduate enzymology, when I was a student at least! These enzymes contain a catalytic triad (Histidine, Aspartate and Serine), in a constellation that promotes the hydrolysis of the peptide bond. The structural properties of the R group of the amino acid side chain such as Lysine or Arginine (in the case of Trypsin) provide the "specificity" determinant for directing the hydrolysis reaction. Some of the most interesting early experiments using site directed mutagenesis were carried out on the serine proteases and this helped us to develop a more robust understanding of some of the general features of enzyme catalysis (you can read a recent review of the early work and more recent experiments here). The combination of these three amino acids in creating an O- (or oxyanion) to facilitate hydrolysis coupled with the "attachment" to a distinctive chemical group (such as the long positively charged side chains of Lysine or Arginine), is an important theme in our understanding of hydrolytic enzymes that act on biopolymers. [Can you think of an analogy in the field of nucleic acids?] In vivo, proteases can serve many roles: from nutritional, like Trypsin, to regulatory (in the cell cycle) to recycling of amino acids via the proteosome. 

Finally, making peptide bonds. So far this has been a proteo-centric Blog. But just think for a minute about the origins of Life (at the molecular level). Proteins are pretty complex molecules and enzymes have taken years of evolutionary time to settle into their catalytically efficient sequences and shapes. The determination of the structure of the Ribosome, together with earlier work by geneticists and biochemists, revealed that the catalytic centre of peptide bond synthesis is made of RNA, not protein! The task of producing crystals of the quality required to determine the high resolution structure of the Ribosome had been a labour of love for many groups, for many years. However finally in around 2000, the first high resolution structures appeared (see here). The award of the NP to Steitz, Yonath and Ramakrishnan was just recognition (in my view) for the tenacity of their research teams over many years in providing us with a working template for understanding the molecular stages of protein synthesis and the mode of action of a number of antibiotics. The condensation of an amino acid with the growing polypeptide chain on the Ribosome is where nucleic acid and protein chemistry converge. The flow of information from the digitally encoded genes through to the functionality of proteins is at the heart of all Life on Earth. Anyone with an interest in Life Sciences must take time to understand the chemistry underpinning, what Francis Crick called the "central dogma" of Molecular Biology. [Before I finish, it would be remiss of me not to mention the reverse flow of information that was discovered in viruses through their recruitment of Reverse Transcriptases, but that's for another time!]

1 comment:

  1. prof premraj pushpakaran writes -- 2017 marks the centenary year of John Cowdery Kendrew!!!