Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 38

Table 3.6 Plasma Hcy-thiolactone levels are elevated in human genetic hyperhomocysteinemia
(Data from [93])

Hcy-thiolactone (nM) Hcy ( μ M)

Patient genotype (n) Mean SD Range Mean SD Range

Unaffected (9) 0.2 0.14 0.1-0.4 7.2 0.9 6.0-8.6

CBS / (14)

14.4 30.4 0.1-100.8 36.1 25.8 15-93

MTHFR / (4) 11.8 8.8 2.9-22.2 50.1 15.1 23-68

MTHFR +/ (6)

0.5 0.3 0.1-1.0 7.8 2.8 5.2-12.2

the synthetic/editing active site [238], whose major function is to carry out the
synthesis of methionyl-tRNA for protein biosynthesis [239]. Whether methionine
or Hcy completes the synthetic or editing pathway, respectively, is determined by
the partitioning of its side chain between the specificity and thiol-binding subsites
of the synthetic/editing active site [240]. A subsite that binds carboxyl and -amino

groups of methionine or Hcy does not appear to contribute to specificity [238].
Methionine completes the synthetic pathway because its side chain is firmly bound
by the hydrophobic and hydrogen bonding interactions with the specificity subsite
(Fig. 3.8 , top panel).
The crystal structure of E. coli MetRS•Met complex reveals that hydrophobic
interactions involve side chains of Tyr15, Trp253, Pro257, and Tyr260; Trp305
closes the bottom of the hydrophobic pocket but is not in the contact with the
methyl group of the substrate methionine. The sulfur atom of the substrate methio-
nine makes two hydrogen bonds: one with the hydroxyl of Tyr260 and the other
with the backbone amide of Leu13 [239]. Crystal structures of E. coli MetRS
complexed with analogs of methionine and methionyl adenylate show that residues
Tyr15 and Trp253 play key roles in the strength of the binding of methionine and of
its analogs and thus determine the specificity of the enzyme for methionine. Full
motions of these residues are required to recover the maximum in free energy of
binding: in the closed conformation (MetRS•Met complex), Tyr15 interacts with
the amino nitrogen of the methionine substrate by forming a

α

bond, and Trp253
maintains hydrophobic interaction with methionine (assisted by a

π - π

bond
between Trp253 and Phe300) [241], whereas in the open conformation (free
MetRS), Tyr15 and Trp253 flip over to the other side of the active site pocket.
Residue Tyr15 also controls the size of the hydrophobic pocket where the amino
acid side chain interacts. In addition, His301 appears to participate to the specific
recognition of the sulfur atom of methionine [242].
The active site residues Trp253 and Tyr15 that control the conformational
flexibility of MetRS are highly conserved in the MetRS sequence of all species,

π - π

Fig. 3.7 (continued) hydrolysis from dithiothreitol (DTT)-treated cellular (e) and extracellular (f)
proteins from HUVEC cultures labeled with [ 35 S]Hcy are shown. N-linked Hcy liberated from
proteins during acid hydrolysis is converted to Hcy-thiolactone (HcyT in the figure) (Reproduced
from [74] and [139])