Enzyme tyrosyl-tRNA synthetase

Figure 4.15 Schematic diagram of the enzyme tyrosyl-tRNA synthetase, which couples tyrosine to its cognate transfer RNA. The central region of the catalytic domain (red and green) is an open twisted a/p stmcture with five parallel p strands. The active site is formed by the loops from the carboxy ends of P strands 2 and S. These two adjacent strands are connected to a helices on opposite sides of the P sheet.

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]...

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.

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. 

Chemical reactivity and hydrogen bonding 320 Proton-transfer behaviour 321 Intramolecular hydrogen-bond catalysis 344 Enzyme catalysis and hydrogen bonding 354 Chymotrypsin 354 Thermolysin 355 Carboxypeptidase 355 Tyrosyl tRNA synthetase 356 Summary 366 Acknowledgements 367 References 367... 

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... 

In addition to its usefulness in probing the role of individual residues in catalysis, site-directed mutagenesis has proved invaluable in dissecting the gross nature of enzymes. The tyrosyl-tRNA synthetase has some interesting properties... 

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. 

These effects are nicely illustrated by changes at position 51.20,21 The tyrosyl-tRNA synthetase from B. stearothermophilus has Thr at this position. The tyrosyl-tRNA synthetase from Bacillus caldotenax differs by just four amino acid residues from that from B. stearothermophilus, but one of the changes is an Ala at position 51 23 The enzyme from E. coli is only 50% identical in sequence but has Pro at... 

In the case of oligomeric proteins in which subunit contact regions have been revealed by X-ray crystallography34 353 or other methods described above,363 the equilibrium between oligomer and monomer can be changed by site-directed mutagenesis. For example, stable monomers of tyrosyl-tRNA synthetase were produced by a mutation of Phe-164 at the subunit interface to Asp, and it was revealed that the monomers are inactive and do not bind the substrate tyrosine.343 In the case of yeast triosephosphate isomerase, replacement of Asn-78 at the subunit interface did not cause dissociation of subunits under normal conditions.353 However, the stability of the enzyme was significantly lowered by the mutation, probably due to decreased subunit-subunit interaction.353... 

Energy of individual hydrogen bonds in a protein tyrosyl-tRNA synthetase as a test case. When an enzyme interacts with its substrate, the bound water molecules... 

The use of the EVB and Equation (13) in studies of reactions in solutions has been extended to studies of LFERs in enzymes. The successes of this approach have been demonstrated in studies of carbonic anhydrase,46 p21 Ras,44,47 and tyrosyl-tRNA synthetase.45 At present, we view these studies as the most quantitative LFER studies of enzymes. It is also useful to point out the successes of our approach in LFER studies of electron transport in proteins (e.g., Ref. 48). 

Warshel, A., Schweins, T. and Fothergill, M. (1994). Linear free energy relationships in enzymes. Theoretical analysis of the reaction of tyrosyl-tRNA synthetase. J. Am. Chem. Soc. 116, 8437-8442... 

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