Subject serine

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Glycogen synthase also exists in two distinct forms which can be interconverted by the action of specific enzymes active, dephosphorylated glycogen synthase I (glucose-6-P-independent) and less active phosphorylated glycogen synthase D (glucose-6-P-dependent). The nature of phosphorylation is more complex with glycogen synthase. As many as nine serine residues on the enzyme appear to be subject to phosphorylation, each site s phosphorylation having some effect on enzyme activity. 

In addition, eNOS is subject to protein phosphorylation. It can be phosphotylated on several serine (Ser), threonine (Thr), and tyrosine (Tyr) residues however, major changes in enzyme function have been reported for the phosphorylation of amino acid residues Seri 177 and Thr495 (in the human eNOS sequence) (Fig. 3). 

TRAM is subject to control through phosphorylation by protein kinase C-e. It is phosphorylated on serine 16 which is located close to the myristoylation site which is TRAM cannot signal without this phosphorylation or if the myristoylation site has been mutated. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

On intuition, a minute amount of water was added to the solvent (ethyl acetate) in the first crystallization experiment containing a molar excess of imidazole corresponding to 1, Regularly shaped crystals were formed within one hour. Such a crystal, subjected to X-ray analysis, has the structure as shown in Fig. 41 U1). Apart from the formation of the expected salt-type associate (carboxylate-imidazolium ion pair, cf. Sect. 4.2.2), two water molecules are present in the asymmetric unit of the crystal structure. This fact called our attention again to the family of serine protease enzymes, where water molecules are reported as being located in the close vicinity of the active sites 115-120),... 

The phosphonium and carbenium salts are efficient reagents for activating and coupling A-alkoxycarbonylamino acids as well as peptide acids. However, the requirement for tertiary amine to effect the reaction has several implications. The base renders hydroxyl groups subject to acylation. Hence, the side chains of serine and threonine and any hydroxymethyl groups of a resin that have not been derivatized... 

Only three amino acids have a hydroxyl functional group in their side chain tyrosine, serine and threonine. Some kinases target only tyrosine residues (tyrosine kinases) whereas others may phosphorylate serine or threonine (Ser/Thr kinases). An enzyme protein (the substrate for the kinase) may have several tyrosine, serine or threonine residues within its primary sequence, but only some of these are subject to phosphorylation by a particular kinase (see Figure 3.6)... 

Glycogen phosphorylase isoenzymes have been isolated from liver, brain and skeletal muscle. All forms are subject to covalent control with conversion of the inactive forms (GP-b) to the active forms (GP-a) by phosphorylation on specific serine residues. This phosphorylation step, mediated by the enzyme phosphorylase kinase, is initiated by glucagon stimulation of the hepatocyte. Indeed, the same cAMP cascade which inhibits glycogen synthesis simultaneously stimulates glycogenolysis, giving us an excellent example of reciprocal control. 

The peptidases were separated into catalytic types according to the chemical nature of the group responsible for catalysis. The major catalytic types are, thus, Serine (and the related Threonine), Cysteine, Aspartic, Metallo, and As-Yet-Unclassified. An in-depth presentation of catalytic sites and mechanisms, based on this classification, is the subject of Chapt. 3. 

A true appreciation of the subtle and complex ways in which the nucleosome can influence gene expression, has come only recently, largely through studies of the post-translational modifications to which all histones are subject and of the enzymes that add and remove these modifications. It has been known for many years that the histone N-terminal tails are exposed on the surface of the nucleosome and that selected amino acid residues are subject to a variety of enzyme-catalyzed, post-translational modifications. These include acetylation of lysines, phosphorylation of serines, and methylation of lysines and arginines ([6,7], see also chapters by Davie, and Ausio and Abbott, this volume). The locations of the histone N-terminal tails in the nucleosome and the residues that can be modified are shown in Fig. 1. 

In cells of the mammary gland, either in normal epithelial or in cancerous cells, the packaging of chromosomal DNA into chromatin restricts the access of the transcription machinery, thereby causing transcriptional repression. The basic N-termini of histones are subject to post-translational modifications, including lysine acetylation, lysine and arginine methylation, serine phosphorylation and ubiquitinylation [56]. It has been proposed in the histone code hypothesis that the intricate pattern of modifications of the N-terminal histone tail influences gene regulation [57]. 

The synthesis of lanthionines via dehydroalanines has been carried out using an (9,5-di-benzoylated Z-L-Cys-L-Ser-OMe. 31 Using (3-elimination under basic conditions in sodium methoxide and subsequent intramolecular Michael addition, the corresponding L-lanthio-nine derivative was formed. However, as a final step the cyclolanthionyl dipeptide would have to be cleaved at the amide bond to obtain lanthionine. The same authors reported the use of the O-tosylated serine dipeptide which was subjected to (3-elimination, forming the desired dehydroalanine derivative. However, this synthesis also yielded cyclic L-lanthionine dimers as a result of an intermolecular Michael addition. The amounts of cyclolanthionine and lanthionine dimer derivatives depended on the initial concentration of the dipeptides. In addition, the directed synthesis of cyclic lanthionine dimers, which were produced from N-benzyloxycarbonyl-W-trityllanthionine monomethyl ester, has been reported. 32 ... 

Phosphorylation of serine or threonine residues involves an ATP-dependent addition of a phosphate group to a primary (serine) or secondary (threonine) alcohol. Phos-phorylated proteins are often subject to rapid degradation. 

In glycoproteins containing a carbohydrate-L-serine linkage, such as 50, or a carbohydrate-L-threonine linkage, the carbohydrate is released from tire protein on treatment with base. The carbohydrate released is then subjected to further degradation, until an alkali-stable structure is formed. This reaction was used by Kabat, Lloyd,... 

---