Tyrosyl adenylate

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.

Figure 4.18 Side chains of the tyrosyl-tRNA synthetase that form hydrogen bonds to tyrosyl adenylate. Green residues are from p strand 2 and the following loop regions, yellow residues are from the loop after P strand S, and brown residues are from the a helix before P strand S. (Adapted from T. Wells and A. Fersht, Nature 316 656-657, 1985.)...

Brick, R, Bhat, T.N., Blow, D.M. Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate. /. Mol. Biol. 208 83-98, 1988. 

The structure of the complex between native enzyme and tyrosyl adenylate has been elucidated (Rubin and Blow, 1981). Tyrosyl adenylate is one of the products of the first step in the enzyme mechanism (47), and is the substrate for the second step of the reaction (48). Eleven possible hydrogen bonds are identified and the structure is given in [88]. Previously, the structure of the complex between the enzyme and the competitive inhibitor tyrosinyl adenylate had been clarified (Monteilhet and Blow, 1978). 

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. 

The tyrosyl-tRNA synthetase alters the equilibrium constant for the formation of tyrosyl-adenylate by a factor of 107 by a strain mechanism10 (Chapter 15, section I). 

There is no doubt that the enzyme-bound aminoacyl adenylate is formed in the absence of tRNA. It may be isolated by chromatography and the free aminoacyl adenylate obtained by precipitation of the enzyme with acid.47 48 Furthermore, the isolated complex will transfer its amino acid to tRNA. The crystal structure of the tyrosyl-tRNA synthetase bound to tyrosyl adenylate has been solved (Chapter 15, section B). 

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... 

A high-resolution structure of a native enzyme is an admirable basis for any mechanistic study relating activity to precise details of structure. It is even better when structures of complexes with substrates and intermediates are available, as is the case with the tyrosyl-tRNA synthetase and tyrosyl adenylate (Figure 15.1). The E Tyr-AMP complex has two remarkable features. The first is the absence of groups that are candidates for roles in classical catalysis. The second is the... 

The catalytic asymmetry of heterodimers was used to show that the wild-type enzyme is asymmetrical in solution. The enzyme is frozen into two populations 50% are active in one subunit and 50% in the other. For example (Figure 15.20), heterodimers containing Asn-45 on one subunit form 0.5 mol of Tyr-AMP per mole of dimer rapidly at wild-type rate (ty2 20 ms) and a further 0.5 mol four orders of magnitude more slowly ( 1/2 = 200 s) at the rate expected for mutant. If the half-of-the-sites activity were induced by the formation of the first mole of tyrosyl adenylate, then the wild-type site would be the one that is occupied. Because this does not happen, there must be frozen-in asymmetry that is randomly distributed between active wild-type subunit/inactive mutant and inactive wild-type site/active mutant. Any interconversion of active and inactive subunits is on a much slower time scale than the half-life of several minutes required for formation of E Tyr-AMP at the mutated site (Asn-45). There is no evidence that the behavior of the heterodimers is different from that of wild-type enzyme. 

A related phenomenon is half-of-the-sites or half-site reactivity, by which an enzyme containing 2n sites reacts (rapidly) at only n of them (Table 10.2). This can be detected only by pre-steady state kinetics. The tyrosyl-tRNA synthetase provides a good example, in that it forms 1 mol of enzyme-bound tyrosyl adenylate with a rate constant of 18 s1, but the second site reacts 104 times more slowly.13 However, as will be seen in Chapter 15, section J2b, protein engineering studies on the tyrosyl-tRNA synthetase unmasked a pre-existing asymmetry of the enzyme in solution. 

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... 

Figure 15.1 Residues of the tyrosyl-tRNA synthetase that form hydrogen bonds with tyrosyl adenylate.

Figure 15.13 LFERs for the formation of tyrosyl adenylate, (a) logfc3 versus logk3/k-3 (b) log off versus Kdiss (c) log (k3/KTK A) verses log off (d) log (k3/KTK A) versus logA diss. (Data from A. R. Fersht, R. J. Leatherbarrow, and T. N. C. Wells, Biochemistry 26, 6030 (1987) and T. N. C. Wells and A. R. Fersht, Biochemistry 28,9201 (1989).)...

Aminoacyl adenylates have long been known to be high energy compounds, but their free energies of hydrolysis had not been accurately measured. This was accomplished for tyrosyl adenylate using the Haldane approach (Chapter 3, section H) and mutants of the tyrosyl-tRNA synthetase. The equilibrium constant for the formation of tyrosyl adenylate in solution (Absolution) = [Tyr-AMP] [PPi]/ [Tyr] [ATP]) is related to the rate and equilibrium constants for the enzymatic reaction illustrated in Figure 15.21 by equation 15.8. 

Our understanding of synthase reactions and the types of the active sites involved in these reactions was advanced substantially by the crystallization and structural solution of tyrosyl-tRNA synthase complexed with the reaction intermediate tyrosyl-adenylate (fig. 29.10). The reaction intermediate is bound in a deep cleft in the enzyme and interacts with it through 11 hydrogen bonds. Six of these bonds are with the AMP moiety, and five are with the tyrosyl moiety of the intermediate. The amino acid selectivity of tyrosyl-tRNA synthase is thus determined primarily by the formation of specific hydrogen bonds with the amino acid. 

Fig. 19.20. Schematic drawing of hydrogen-bonding interactions between the enzyme tyrosyl-tRNA synthetase and the substrate analog tyrosyl adenylate. The enzyme groups interacting with tyrosyl adenylate are boxed, MC indicates main-chain C=0 or N-H groups [647]...

Brown et al. (1987) compared the structures of wild-type tyrosine— tRNA ligase (tyrosyl-tRNA synthetase) and the Thr-5 l Pro-51 mutant. The stronger affinity of the mutant for tyrosyl—adenylate complexes was attributed to the absence for the mutant of an unfavorable desolvation of residue 51. 

Figure 3-3. Hydrogen bonds between the tyrosyl-tRNA synthetase and tyrosyl adenylate.

The tyrosyl adenylate (Tyr-AMP) is formed and then held tightly at the active site until it reacts with tRNA in the second step. The equilibrium for the formation of Tyr-AMP is unfavorable in solution (ATeq = 3.5 x 10 ) but is near unity at the active site (/(eq = 2.3). Thus, the enzyme must contribute 9.3 kcal/mol toward stabilization of the products at the active site. This is accomplished by the increasing strength and number of hydrogen-bonding interactions between the reactants and active site amino acids as the reaction proceeds. 

Fio. 10. Gibbs free energy profiles for the formation of tyrosyl adenylate and pyrophosphate, as defined in Eq. (2), by wild-type (energy levels in dashed lines) and mutant (energy levels in solid lines) tyrosyl-tRNA synthetases, using standard states of 1 W for tyrosine, ATP, and pyrophosphate. [Reprinted with permission from Ref. (25/).]... 

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