Tyrosine adenylation

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

Figure 4.16 A schematic view of the active site of tyrosyl-tRNA synthetase. Tyrosyl adenylate, the product of the first reaction catalyzed by the enzyme, is bound to two loop regions residues 38-47, which form the loop after p strand 2, and residues 190-193, which form the loop after P strand 5. The tyrosine and adenylate moieties are bound on opposite sides of the P sheet outside the catboxy ends of P strands 2 and 5.

Scheme 10.8 Biosynthesis of epothilone. Individual PKS domains are represented as circles and individual NRPS domains as hexagons. Acyl carrier proteins (ACPs) and thiola-tion domains (T) are posttranslationally modified by a phos-phopantetheinyl group to which the biosynthetic intermediates are covalently bound throughout the chain assembly. The thioesterase domain (TE) cyclizes the fully assembled carbon chain to give the 16-membered lactone. Following dehydration of Cl 2—Cl 3 to give epothilones C and D, the final step in epothilone biosynthesis is the epoxidation of the C12=C13 double bond by the cytochrome P450 enzyme P450epol<. KS ketosyn-thase KS(Y) active-site tyrosine mutant of KS AT acyltransfer-ase C condensation domain A adenylation domain ...

Some signal pathways that activate the adenylate cyclase phosphorylate tyrosine residues (Y535) of the TAF2 domain, which, as previously mentioned, is dependent on hormone binding. In this case, modulation of the transcription... 

Note that these structures are related to that for the amino acid tyrosine, from which they are derived. The adrenal glands, small pieces of tissue that ride on top of the kidneys, secrete these hormones. When they activate adrenergic receptors on the surface of muscle cells, adenylate cyclase is activated, increased cAMP results, and the cascade of events in muscle cells is started (figure 17.1). 

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer of Mr2 X 47 316. It catalyzes the aminoacylation of tRNA1 in a two-step reaction. Tyrosine is first activated (equation 15.1) to form a very stable enzyme-bound tyrosyl adenylate complex. Tyrosine is then transferred to tRNA (equation 15.2).6... 

The crystal structures of the E Tyr and E Tyr-AMP complexes have also been solved.8 Although two moles of tyrosine bind to the crystalline enzyme, only one mole binds to the enzyme in solution. Further, only one mole of tyrosyl adenylate is formed rapidly per mole of dimer, and only one mole of tRNA is bound. The enzyme exhibits half-of-the-sites activity (Chapter 10, section C).9... 

Recognition of an adenylylated amino acid by the proper synthase. Shown is adenyl tyrosine bound to the tyrosyl synthase. A network of H bonds not only stabilizes the reaction but also serves to discriminate between different amino acid residues. MC designates main chain (backbone) carbonyl or amino groups participating in hydrogen bonding. 

Activation of neostriatal tyrosine hydroxylase was observed when cyclic AMP was added to high speed supernatants from rat neostriatum (133). Intraventricular injection of dibutyryl cyclic AMP stimulated tyrosine hydroxylation in the neostriatum (134). However, it is still questionable if under physiological conditions this cyclic AMP involvement in the feedback control of tyrosine hydroxylase activity is mediated by presynaptic dopamine receptors or by presynaptic allo-receptors. In addition, if a dopamine sensitive adenylate cyclase is involved in the regulation of neostriatal tyrosine hydroxylase activity it is relevant to know if this adenylate cyclase is linked to a D-1 and/or a D-2 receptor. At this point in time experimental data are not in favour of the presence of a D-l receptor linked to an adeiylate cyclase on the varicosities of dopaminergic neurons in the neostriatum. E.g. concentrations of dopamine agonists stimulating cyclic AMP formation inhibit tyrosine... 

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