Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Biosynthesis
Post-translational Modification of Proteins

At the fifth and final stage of Protein Synthesis, the Tertiary Structure of the polypeptide molecule is formed and Processing takes place. Synthesized on the ribosome in strict accordance with the genetic program, the linear, one-dimensional polypeptide molecule already contains specific information. Such a molecule is referred to as conformational; that is, rather than undergoing chaotic structural changes, it is transformed (processed) into a strictly defined three-dimensional entity that possesses its own—now functional—information. This principle holds true for Proteins that primarily perform structural Functions, but not for biologically inactive protein precursors whose functional activity emerges later As a result of diverse transformations collectively known as "post-synthetic or post-translational modification." Such modifications of the polypeptide structure begin either immediately after Translation or even before the tertiary STRUCTURE OF THE protein molecule has fully formed.

In addition to the aforementioned proteolytic removal of the signal peptide, the initial N-terminal Methionine is cleaved off in many proteins. Prokaryotic Cells have been shown to contain specialized Enzymes that modify N-terminal residues—specifically, deformylase, which catalyzes the removal of the formyl group from N-terminal methionine, and aminopeptidases, which catalyze the Cleavage of not only N-terminal formylmethionine (or methionine in eukaryotes), but potentially other amino acid residues from the N-terminus of the peptide as well. Similar limited post-synthetic proteolysis affects certain proproteins or proenzymes (such as trypsinogen and chymotrypsinogen) and hormone precursors (such as preproinsulin and pre-β-lipotropin). In some cases, C-terminal Modification of the synthesized protein is also observed.

As is well known, the segment of DNA that carries the information for the synthesis of an individual protein is called a Gene, whereas the segment that controls and is responsible for the synthesis of a single polypeptide chain is termed a Cistron. Consequently, if a protein consists of multiple (more than one) Polypeptides, one would naturally expect multiple (more than one) cistrons to participate in its synthesis. This is not always the case, particularly when The polypeptide chains are identical (for example, the a₂ and ß₂ chains of Hemoglobin). Furthermore, if the peptide chains of a given protein molecule are non-identical, it does not necessarily mean they are synthesized as a result of distinct cistrons. Such a protein may instead be synthesized as a single polypeptide chain followed by proteolytic cleavage at one or more sites, with the subsequent removal of inactive segments. A typical example of this type of modification is the hormone Insulin, which is synthesized as a single polypeptide precursor, preproinsulin. Upon Enzymatic Hydrolysis, this precursor is first converted into the inactive intermediate proinsulin and then into the active insulin hormone, which consists of two polypeptide chains of different sizes and sequences (see Fig. 1.14).

It should be emphasized, however, that post-translational Chemical modification of proteins involving the side chains of individual Amino Acids accounts for a significantly larger share of modifications. One such crucial modification is the covalent attachment of a prosthetic group to the protein molecule. For example, the biologically active three-dimensional configuration of aminotransferases—enzymes that catalyze AMINO ACID Transamination reactions—is formed only after Pyridoxal phosphate attaches to the ε-amino group of a Lysine residue within the protein moiety (the apoenzyme). Certain proteins undergo glycosylation by acquiring oligosaccharide residues (yielding Glycoproteins), thereby ensuring their targeted delivery to recipient cells. Chemical modifications of proteins are also widely prevalent through the hydroxylation of Proline and lysine residues (during Collagen molecule formation), methylation (of lysine and glutamate residues), Acetylation of various N-terminal amino acids, and carboxylation of glutamate and aspartate residues in A number of proteins (via The addition of an extra carboxyl group). In particular, prothrombin (a protein of the Blood Coagulation

system) contains several Y-carboxyglutamate residues at its N-terminus, in The formation of which a vitamin K-dependent enzyme plays an active role. It is hypothesized that y-carboxyglutamate residues participate in binding the Ca²⁺ ions required to initiate blood clotting.

One of the most widespread post-synthetic chemical modifications is the phosphorylation of Serine and Threonine residues, which occurs, for example, in histone and non-histone proteins as well as milk casein. The phosphorylation-dephosphorylation of serine OH-groups is absolutely essential for numerous enzymes, such as Glycogen phosphorylase and glycogen synthase. Furthermore, the phosphorylation of specific Tyrosine residues within a protein molecule is currently regarded as a potential specific stage in the formation of oncoproteins during the malignant transformation of normal cells. Also well known are the oxidation reactions of two Cysteine residues and the formation of intra- and interchain Disulfide Bonds during tertiary structure formation (folding). This ensures not only protection against external Denaturing Agents, but also the ESTABLISHMENT OF THE native conformation and the manifestation of biological activity.

Less widely known are the farnesylation reactions affecting cysteine residues in several proteins: G protein (see Chapter 8), members of the nuclear matrix protein group, as well as ras oncogene proteins and Proto-oncogenes. The source of the isoprenyl groups is farnesyl pyrophosphate (an intermediate in Cholesterol synthesis). Evidence has emerged that blocking the farnesylation reaction using specific pharmacological agents (inhibitors) leads to the loss of the carcinogenic activity of the ras oncogene. These findings could serve as the basis for developing effective therapies against human tumor diseases based on inhibiting the post-translational modification of proteins in general or oncoproteins in particular.

It should be noted that although METABOLISM/35.html">Protein Biosynthesis—a complex, multi-step process—has been detailed in numerous reviews and monographs, our understanding of the Structural and functional relationships at many of its stages remains insufficient. Indeed, Ribosomes have been isolated and characterized (most thoroughly in E. coli), revealing that they consist of numerous individual proteins and Three types of RNA molecules; furthermore, the Amino Acid Sequence of all 55 ribosomal protein molecules as well as the Primary and secondary structures of the three RNA types have been elucidated, and the three-dimensional structure of individual prokaryotic ribosomal proteins is being intensively studied. Nevertheless, many essential details of the protein synthesis mechanism remain unclear. For instance, it is still not fully understood which regions or Components of the ribosomes are responsible for the initiation, elongation, and termination of protein synthesis, nor is the Molecular Mechanism of translocation and peptidyl transferase reactions entirely clear, nor the subtle interactions of ribosomes with protein factors, mRNA, tRNA, and Antibiotics. Considerable effort will still be required to map the complete molecular architecture of ribosomes and their individual subparticles, as well as to clarify and obtain precise data on their tertiary structure, shape, and dimensions sufficient to decode at THE MOLECULAR LEVEL the ribosome's function in the complex process of protein synthesis.

Non-ribosomal Peptide Synthesis. Accumulated data indeed indicate that the template mechanism of synthesis underlies The biosynthesis of nearly all proteins in living organisms. Nevertheless, the synthesis of certain low-molecular-weight (short) Peptides in biological systems can proceed not only without the participation of Nucleic Acids—specifically without template mRNA—but even in the absence of ribosomes. As early as the 10th International Congress of Biochemistry in Hamburg in 1976, F. Lipmann (USA) and K. Kurahashi (Japan) presented experimental evidence demonstrating the synthesis of two natural cyclic peptide antibiotics, gramicidin S and tyrocidine, both in whole extracts derived from Bacillus brevis and in protein fractions isolated from these extracts. Specifically, two purified protein preparations isolated from B. brevis extracts ensured the precise assembly of the cyclic polypeptide gramicidin S, which consists of 10 amino acid residues arranged in a strict sequence. The purified protein fractions (with molecular weights of 100,000 and 180,000) required only the presence of free amino acids, ATP, and Mg²⁺ ions to synthesize this cyclic decapeptide ((D-phenylalanyl-prolyl-valyl-ornithyl-leucine)₂):

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It has been demonstrated that the light protein fraction (molecular weight 100,000) is responsible for the racemization and incorporation of D-phenylalanine into the nascent polypeptide chain, whereas the heavy fraction (molecular weight 180,000) governs the incorporation of the remaining four L-amino acids; both enzymes also participate in peptide bond formation. Similarly, an identical pentapeptide is synthesized on an adjacent multienzyme complex; the two pentapeptides are subsequently joined in a HEAD-to-tail fashion with chain closure, yielding the cyclic decapeptide. The synthesis mechanism is thought to involve the preliminary formation of aminoacyl adenylates (mediated by these same enzymes), from which The amino acid residues are then transferred to the SH groups of both enzymes. This process generates activated intermediate thioesters analogous to those involved in the synthesis of Higher Fatty acids (see Chapter 11). A covalently linked phosphopantetheine residue has been discovered in The structure of the first (light) enzyme, leading researchers to hypothesize that its thiol group participates in transferring the growing peptide chain from one catalytic site on the enzyme to another. A comparable mechanism of synthesis has also been proven for the antibiotic tyrocidine (a decapeptide) and for the 13-membered cyclic peptide antibiotic myacubacillin [mycobacillin].

Thus, nature (figuratively represented by the bacterial Cell) apparently has not entirely discarded the atavistic mechanism of protein body synthesis that existed prior to the template, ribosomal pathway, and continues to employ quite primitive yet sufficiently efficient techniques for this purpose.