Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

Journal Article · Fri Feb 25 04:00:00 EST 2011 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2011.01.080· OSTI ID:22204801

Jomrit, Juntratip ^[1]; Summpunn, Pijug ^[1]; Meevootisom, Vithaya ^[2]; Wiyakrutta, Suthep ^[2]

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1. Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400 (Thailand)
2. Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400 (Thailand)

Research highlights: {yields} Stereochemical mechanism of PLP enzymes is important but difficult to determine. {yields} This new method is significantly less complicated than the previous ones. {yields} This assay is as sensitive as the radioactive based method. {yields} LC-MS/MS positively identify the analyte coenzyme. {yields} The method can be used with enzyme whose apo form is unstable. -- Abstract: A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in {sup 2}H{sub 2}O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-{sup 2}H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The {sup 2}H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the {sup 2}H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of {sup 2}H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

• OSTI ID: 22204801
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 4 Vol. 405; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AMINO ACIDS
AMINOTRANSFERASES
COENZYMES
DEUTERIUM
HYDROGEN TRANSFER
MOLECULAR WEIGHT
PHOSPHATES
PYRIDOXAL
SENSITIVITY
STEREOCHEMISTRY
SUBSTRATES