Ser residues

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

The catalytic triad consists of the side chains of Asp, His, and Ser close to each other. The Ser residue is reactive and forms a covalent bond with the substrate, thereby providing a specific pathway for the reaction. His has a dual role first, it accepts a proton from Ser to facilitate formation of the covalent bond and, second, it stabilizes the negatively charged transition state. The proton is subsequently transferred to the N atom of the leaving group. Mutations of either of these two residues decrease the catalytic rate by a factor of 10 because they abolish the specific reaction pathway. Asp, by stabilizing the positive charge of His, contributes a rate enhancement of 10. ... 

These signals in the NOE spectra therefore in principle make it possible to determine which fingerprint in the COSY spectrum comes from a residue adjacent to the one previously identified. For example, in the case of the lac-repressor fragment the specific Ser residue that was identified from the COSY spectrum was shown in the NOE spectrum to interact with a His residue, which in turn interacted with a Val residue. Comparison with the known amino acid sequence revealed that the tripeptide Ser-His-Val occurred only once, for residues 28-30. 

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Fig. 6. Sequence comparisons of Rieske proteins from spinach chloroplasts, beef heart mitochondria, green sulfur bacteria, and firmicutes. The extended insertion of proteobacterial Rieske proteins as compared to the mitochondrial one is indicated by a dotted arrow. The redox-potential-influencing Ser residue is marked by a vertical arrow. The top and the bottom sequence numberings refer to the spinach and bovine proteins, respectively. Fully conserved residues are marked by dark shading, whereas the residues conserved in the b6f-group are denoted by lighter shading.

An abasic peptide (Ac-Gys -Lys-(Ser-Ala-Ala-Lys)4-Ser-Gly-Lys-NH2) with unmodified Ser residues was synthesized and used as a control. No induced cooperative binding to. TAjGsAsT) was observed in the UV melting curve with this abasic peptide. This indicated that the cooperative melting between aPNA and its complementary DNA was not merely a reflection of nonspecific interactions be-... 

The replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence obtained by Cooke et al. [13] and by Rexova-Benkova et aL [14] that a histidine readue is critical for the activity of the enzyme. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzyme activity to 48% and 6% reqrectively, indicating that Ser residues are also inqrortant for the activity. 

The finding of proteasomal degradation of type 2 ACS isozymes via Etol interaction raises the question whether there exists a modification for blocking this process. The most promising candidate for such a modification is phosphorylation of C-terminus. Prohahly the phosphorylation of the Ser residue (underlined)... 

Desferrimaduraferrin is a Fe " complexing metabolite of Actinomadura madurae 185). It consists of salicylic acid, p-Ala, Gly, L-Ser and 77 -hydroxy-77 -methyl-L-Om, with the latter incorporated in a heterocyclic system (Fig. 4, 13). From the same species the madurastatin group was obtained 136). The main representative A1 shows the sequence salicylic acid, o-azaridine carboxylic acid, L-Ala, p-Ala, 77 -hydroxy-77 -methyl-Om, L-cOHOm (Fig. 4, 14). In A2 the azaridine ring is opened giving a Ser residue, A3 is an isomer of the open form with the salicylic acid bound to the hydroxy group of Ser. B1 and B2 are the precursors A-salicyloyl-azaridine carboxylic acid and A-salicyloyl-Ser. The madurastatin species A1 forms a 1 1 Fe " complex as shown by mass spectrometry. 

The pair of lysine residues that serve as ubiquitination sites in both molecules are marked by vertical lines. Two overlapping acidic residues are marked by -I-. Ser residues are larger and bolded, whereas all acidic residues are bolded and italicized (6 in IkBo and 5 in pl05). 

Lipases belong to the subclass of serine hydrolases, and their structure and reaction mechanism are well understood. Their common a/p-hydrolase enzyme fold is characterized by an a-helix that is connected with a sharp turn, referred to as the nucleophilic elbow, to the middle of a P-sheet array. All lipases possess an identical catalytic triad consisting of an Asp or Gin residue, a His and a nucleophilic Ser [14]. The latter residue is located at the nucleophilic elbow and is found in the middle of the highly conserved Gly—AAl—Ser—AA2—Gly sequence in which amino acids AAl and AA2 can vary. The His residue is spatially located at one side of the Ser residue, whereas at the opposite side of the Ser a negative charge can be stabilized in the so-called oxyanion hole by a series of hydrogen bond interactions. The catalytic mechanism of the class of a/P-hydrolases is briefly discussed below using CALB as a typical example, since this is the most commonly applied lipase in polymerization reactions [15]. 

In lipase-catalyzed ROP, it is generally accepted that the monomer activation proceeds via the formation of an acyl-enzyme intermediate by reaction of the Ser residue with the lactone, rendering the carbonyl more prone to nucleophilic attack (Fig. 3) [60-64, 94]. Initiation of the polymerization occurs by deacylation of the acyl-enzyme intermediate by an appropriate nucleophile such as water or an alcohol to produce the corresponding co-hydroxycarboxylic acid or ester. Propagation, on the other hand, occurs by deacylation of the acyl-enzyme intermediate by the terminal hydroxyl group of the growing polymer chain to produce a polymer chain that is elongated by one monomer unit. 

The large subimit of RNA polymerase II plays an important role at the beginning of the transcription process. The large subimit of the mammalian enzyme contains 52 copies of the heptamer sequence YSPTSPS in the C-terminal domain (CTD) at which phosphorylation occurs. Phosphorylation occurs extensively on the Ser-residues of the CTD, to a lesser degree at the Thr-residues, and, very rarely, at the Tyr-residues. Two forms of RNA polymerase II can be isolated from cellular extracts a underphosphory-lated form and a hyper-phosphorylated form. The isoforms fulfill different functions RNA polymerase found in the initiation complex tends to display little or no phosphorylation at the C-terminus of the large subunit, while RNA polymerase II active in elongation is hyperphosphorylated in this region of the protein. 

Fig. 2.10. Change in charge state of proteins via phosphorylation. The phosphorylation of Ser residues is catalyzed by a Ser/Thr-specific kinase that utilizes ATP as the phosphate group donor. The product of the reaction is a Ser-phosphate ester which carries a net charge of -2 at physiological pH.

The membrane-associated Akt kinase is now a substrate for protein kinase PDKl that phosphorylates a specific Thr and Ser residue of Akt kinase. The double phosphorylation converts Akt kinase to the active form. It is assumed that the Akt kinase now dissociates from the membrane and phosphorylates cytosolic substrates such as glycogen synthase kinase, 6-phosphofructo-2-kinase and ribosomal protein S6 kinase, p70 . According to this mechanism, Akt kinase regulates central metabolic pathways of the cell. Furthermore, it has a promoting influence on cell division and an inhibitory influence on programmed cell death, apoptosis. A role in apoptosis is suggested by the observation that a component of the apoptotic program. Bad protein (see Chapter 15) has been identified as a substrate of Akt kinase. 

Structural information on Ser/Thr-specific protein kinases (review Goldsmith and Cobb, 1994, Johnson et al., 1996, Johnson, 1998) indicates a markedly conserved structure of the catalytic domain. In Fig. 7.2a, the structure of the catalytic subimit of protein kinase A is shown in complex with an inhibitor peptide (Knighton et al., 1991). In this case, the inhibitor peptide serves as a model for a phosphorylation substrate. In the form of two Arg residues, it possesses part of a sequence characteristic of phosphorylation sites of protein kinase A, which is defined by two Arg residues in the neighborhood of the Ser residue to be phosphorylated (Fig. 7.3). The inhibitor peptide lacks the... 

Ser residue to be phosphorylated this is replaced by alanine. Due to these characteristics, the inhibitor peptide is bound in a very similar way and with similar affinity to a substrate, but it carmot react and has the property of a pseudosubstrate. The contacts between protein kinase A and a substrate are shown as a model for a peptide known as kemptide, which serves as a phosphorylation substrate. 

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