Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

Abstract

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

The dietary protein paradox and threonine 15N‐depletion: Pyridoxal‐5'‐phosphate enzyme activity as a mechanism for the δ15N trophic level effect

“…This supports the hypothesis of Hare et al . that threonine ammonia‐lyase has an inverse isotope effect, and agrees with the inverse nitrogen equilibrium isotope effects of pyridoxal‐5'‐phosphate cofactor enzymes . While there is probably no single biochemical mechanism that can completely explain the bioaccumulation of 15 N in living organisms, we hypothesize that Schiff base formation between AAs and pyridoxal‐5'‐phosphate enzymes, notably during transamination, plays an integral role in the magnitude and variability of the nitrogen TLE.…”

“…[66] Pyridoxal-5'-phosphate enzymes are known for significant 15 N-equilibrium isotope effects as a result of Schiff base (aldimine) formation. In particular, Abell and O'Leary [67,68] found that glutamate decarboxylase (EC 4.1.1.15) and histidine decarboxylase (EC 4.1.1.22) have large inverse nitrogen equilibrium isotope effects that cause 15 N to be concentrated in the chemical bonds of Schiff bases. This is due to the reversible nature of these reactions, whereby 15 N-amino groups form stronger and more stable carbonnitrogen bonds than 14 N-amino groups.…”

“…The origin(s) for this contradictory finding in terms of our results remains unclear, although it could be related to the in vitro nature of this experiment, the fact that Thr was not chromatographically isolated or self‐described difficulties assocated with the enzyme preparation and isotopic measurement of the substrate in their study. While the mechanism of Schiff base formation between Thr and threonine ammonia‐lyase is similar to that of the enzymes studied by Abell and O'Leary, and thus should yield inverse nitrogen equilibrium isotope effects, further work is needed to directly determine the intrinsic isotope effects of threonine ammonia‐lyase to resolve this issue.…”

Recent Topics in Pyridoxal 5'-Phosphate Enzyme Studies

“…The sequence around PLP-binding lysine (Lys232) showed the conserved sequence Ser-X-His-(P-Pxy)-Lys earlier found in E. coli de carboxylases (32). The carbon and nitrogen isotope effects showed that the overall decarboxylation rate is jointly limited by the rate of transamination and by the intrinsic rate of decarboxylation (171), as is the case for glutamate decarboxylase. The carbon isotope effect of 1.05-1.06 on the decarboxylation step of HisDC is identical to that of GluDC (172), suggesting a similar transition state for the two enzymes.…”

Crystallization of diaminopimelate decarboxylase fromEscherichia coli, a stereospecificD-amino-acid decarboxylase

“…The stereospeci®city of DDCs toward DAP results in inversion of the chiral center (Asada et al, 1981) and inversion may occur with the eukaryotic ODCs. This is in contrast to other bacterial decarboxylases, such as the bacterial ODC, glutamate and histidine decarboxylases, where retention of con®guration is conserved (Abell & O'Leary, 1988;Asada et al, 1984; crystallization papers Yamada & O'Leary, 1978). Here, we report the crystallization of a polyhistidine-tagged DDC from E. coli.…”