Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase

Abstract

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

PhnJ – A novel radical SAM enzyme from the C–P lyase complex

“…To determine the stereospecificity of these transfers, we expressed PhnJ in minimal media that was supplemented with glycine that was specifically deuterated at the proR position. The monodeuterated glycine was made by incubating glycine with the enzyme methionine-γ-lyase (MGL) in the presence of D 2 O (Koulikova et al, 2011). This reaction is illustrated in Scheme 6.…”

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

“…Reaction of Methionine ␥-Lyase with Glycine-It was shown that MGL catalyzes exchange of both C␣-protons of glycine with high stereospecificity for pro-R-proton, which reacts by a factor of 1440 faster than does pro-S-proton (35). In principle, the exchange may proceed through the formation of a quinonoid intermediate or by a concerted mechanism implying fast and reversible transfer of the pro-R-proton from C␣ atom to C4Ј atom or a formation of a six-membered transition state incorporating a water molecule (36) (Scheme 2).…”

“…The rate and the equilibrium constants calculated using Scheme 3 are listed in Table 2. The steady-state rate constant (k ex ) of the isotopic exchange of the two enantiotopic protons of glycine (35) are also presented in Table 2. Having compared the exchange rates with the other rates in Table 2, it was supposed that the complex (E⅐Gly) 1 , which absorbs at 420 nm and is formed in a millisecond time interval, probably corresponds to the external aldimine intermediate with orthogonal orientation of the "right" pro-R-proton (H r ) to the cofactor plane (Fig.…”

Serine 339 in the Catalysis of γ- and β-Elimination Reactions

“…Растворитель удаляли на роторном испарителе. Для определения конфигурации у C-α-атома дейтерированный продукт был превращен в дипептид, L-фенилаланил-[D]-глицин в реакции с Boc-L-Phe-ONp [40,41]. Дипептид растворяли в 0.5 мл D 2 O, и 1 Н-ЯМР-спектр записывали на спектрометре Вruker AMXIII-400 с рабочей частотой 400 МГц.…”