Tài liệu Báo cáo khoa học: The mechanism of a-proton isotope exchange in amino acids catalysed by

The mechanism of a-proton isotope exchange in amino acids catalysed

by tyrosine phenol-lyase

1What is the role of quinonoid intermediates?

Nicolai G. Faleev1

, Tatyana V. Demidkina2

, Marina A. Tsvetikova1

, Robert S. Phillips3 and Igor A. Yamskov1

1

Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia; 2

Engelhardt Institute of

Molecular Biology, Russian Academy of Sciences, Moscow, Russia; 3

Department of Chemistry, Department of Biochemistry and

Molecular Biology, and Center for Metalloenzyme Studies, University of Georgia, Athens, GA, USA

To shed light on the mechanism of isotopic exchange of

a-protons in amino acids catalyzed by pyridoxal phosphate

(PLP)-dependent enzymes, we studied the kinetics of

quinonoid intermediate formation for the reactions of

tyrosine phenol-lyase with L-phenylalanine, L-methionine,

and their a-deuterated analogues in D2O, and we compared

the results with the rates of the isotopic exchange under the

same conditions. We have found that, in the L-phenylalanine

reaction, the internal return of the a-proton is operative, and

allowing for its effect, the exchange rate is accounted for

satisfactorily. Surprisingly, for the reaction with L-methio￾nine, the enzymatic isotope exchange went much faster than

might be predicted from the kinetic data for quinonoid

intermediate formation. This result allows us to suggest the

existence of an alternative, possibly concerted, mechanism of

a-proton exchange.

Keywords: amino acids; isotopic exchange; mechanism;

a-proton; tyrosine phenol-lyase.

Pyridoxal-P-phosphate (PLP)-dependent lyases displaying

broad substrate specificity are able to catalyze stereospecific

isotope exchange of a-protons of various amino acids [1–4]

including both real substrates and reversible competitive

inhibitors, which do not change their chemical identities

under the action of the enzyme. The exchange is usually

performed in heavy water, and proceeds with a complete

retention of the natural (S)-configuration of amino acids.

The characteristic PLP-dependent enzymes in this respect

are tyrosine phenol-lyase (TPL) (EC 4.1.99.2), tryptophan

indole-lyase (EC 4.1.99.1), and L-methionine-c-lyase

(EC 4.4.1.11). These enzymes are used as very effective

biocatalysts for preparation of enantiomerically pure

a-deuterated (S)-amino acids [5–7].

In the framework of the generally accepted notions of

mechanisms of PLP-dependent enzymes the mechanism of

the isotopic exchange traditionally is considered to be

associated with formation of quinonoid intermediates

(Scheme 1). In the holoenzymes (E) the cofactor PLP is

bound in the active site as an
internal aldimine with an

e-amino group of a definite lysine residue. As a result of

interaction with an amino acid substrate, or inhibitor, the

internal aldimine (E) is substituted by an
external one (ES),

which undergoes the abstraction of the a-proton by a

certain enzyme group, leading to formation of a
quinonoid

intermediate (EA). The reversibility of the latter transfor￾mation should lead in heavy water to the isotopic exchange

of the a-proton if the abstracted proton may be easily

exchanged with the solvent. However, the kinetics of

quinonoid formation was examined until now only in water

solutions [8–11], while measurements in heavy water, in

conditions identical to those of the isotopic exchange, were

not performed. No attempts to quantitatively estimate the

rates of the exchange of the abstracted proton in the active

site have been reported. We have noted earlier [8] that no

direct correlation was observed between the amount of the

quinonoid intermediate formed under steady-state condi￾tions in reactions of PLP-dependent enzymes with amino

acids and the rates of the enzymatic isotopic exchange for

the same amino acids.

To answer these questions, we studied in the present work

the kinetics of quinonoid intermediate formation for the

reactions of TPL with L-phenylalanine, L-methionine, and

their a-deuterated analogs in D2O, and compared the results

with the rates of the isotope exchange under the same

conditions. We have found that in the L-phenylalanine

reaction the exchange of the abstracted proton in the active

site proceeds more slowly than the reprotonation reaction,

leading to a considerable internal return of the a-proton.

Allowing for this effect, the rate of the enzymatic isotopic

exchange is accounted for satisfactorily. Surprisingly, for the

reaction with L-methionine the enzymatic isotopic exchange

proceeds much faster than it follows from the kinetic data

for quinonoid intermediate formation. This result allows us

to conclude that the quinonoid is a dead-end complex in this

Correspondence to N. G. Faleev, Nesmeyanov Institute of Organo￾element Compounds, Russian Academy of Sciences, 28 Vavilov Street,

Moscow, 119991, Russia. Fax: +95 1355085, Tel.: +95 1356458,

E-mail: [email protected]

Abbreviations: PLP, pyridoxal-P-phosphate; TPL, tyrosine phenol￾lyase; SOPC, S-o-nitrophenyl-L-cysteine.

Enzymes: tyrosine phenol-lyase (EC 4.1.99.2); tryptophan indole-lyase

(EC 4.1.99.1); L-methionine-c-lyase (EC 4.4.1.11); aspartate amino￾transferase (EC 2.6.1.1).

(Received 6 July 2004, revised 7 September 2004,

accepted 8 October 2004)

Eur. J. Biochem. 271, 4565–4571 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04428.x