PMID- 27235171
OWN - NLM
STAT- MEDLINE
DCOM- 20170531
LR  - 20180207
IS  - 1096-0309 (Electronic)
IS  - 0003-2697 (Linking)
VI  - 507
DP  - 2016 Aug 15
TI  - A continuous spectrophotometric assay and nonlinear kinetic analysis of 
      methionine gamma-lyase catalysis.
PG  - 21-6
LID - S0003-2697(16)30085-9 [pii]
LID - 10.1016/j.ab.2016.05.010 [doi]
AB  - In this article, we present a new, easy-to-implement assay for methionine gamma-lyase 
      (MGL)-catalyzed gamma-elimination reactions of l-methionine and its analogues that 
      produce alpha-ketobutyrate (alpha-KB) as product. The assay employs ultraviolet-visible 
      (UV-Vis) spectrophotometry to continuously monitor the rate of formation of alpha-KB 
      by its absorbance at 315 nm. We also employ a nonlinear data analysis method that 
      obviates the need for an "initial slope" determination, which can introduce 
      errors when the progress curves are nonlinear. The spectrophotometric assay is 
      validated through product analysis by (1)H NMR (nuclear magnetic resonance), 
      which showed that under the conditions of study l-methionine (l-met) and 
      l-methionine sulfone (l-met sulfone) substrates were converted to alpha-KB product 
      with greater than 99% yield. Using this assay method, we determined for the first 
      time the Michaelis-Menten parameters for a recombinant form of MGL from 
      Porphyromonas gingivalis, obtaining respective kcat and Km values of 
      328 +/- 8 min(-1) and 1.2 +/- 0.1 mM for l-met gamma-elimination and 2048 +/- 59 min(-1) 
      and 38 +/- 2 mM for l-met sulfone gamma-elimination reactions. We envisage that this 
      assay method will be useful for determining the activity of MGL gamma-elimination 
      reactions that produce alpha-KB as the end product.
CI  - Copyright (c) 2016 Elsevier Inc. All rights reserved.
FAU - Foo, Timothy C
AU  - Foo TC
AD  - Department of Chemistry and Biochemistry, Florida Atlantic University, Boca 
      Raton, FL 33431, USA.
FAU - Terentis, Andrew C
AU  - Terentis AC
AD  - Department of Chemistry and Biochemistry, Florida Atlantic University, Boca 
      Raton, FL 33431, USA. Electronic address: terentis@fau.edu.
FAU - Venkatachalam, Kallidaikurichi V
AU  - Venkatachalam KV
AD  - Department of Biochemistry, College of Medical Sciences, Nova Southeastern 
      University, Fort Lauderdale, FL 33328, USA. Electronic address: venk@nova.edu.
LA  - eng
PT  - Journal Article
DEP - 20160524
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Butyrates)
RN  - 600-18-0 (alpha-ketobutyric acid)
RN  - AE28F7PNPL (Methionine)
RN  - EC 4.4.- (Carbon-Sulfur Lyases)
RN  - EC 4.4.1.11 (L-methionine gamma-lyase)
SB  - IM
MH  - *Biocatalysis
MH  - Butyrates/chemistry/metabolism
MH  - Carbon-Sulfur Lyases/*analysis/metabolism
MH  - Kinetics
MH  - Methionine/chemistry/metabolism
MH  - Molecular Structure
MH  - Porphyromonas gingivalis/enzymology
MH  - Spectrophotometry, Ultraviolet
OTO - NOTNLM
OT  - Enzyme catalysis
OT  - Methionine gamma-lyase
OT  - Porphyromonas gingivalis
OT  - Pyridoxal-5'-phosphate
OT  - UV-Vis spectrophotometry
EDAT- 2016/05/29 06:00
MHDA- 2017/06/01 06:00
CRDT- 2016/05/29 06:00
PHST- 2016/01/27 00:00 [received]
PHST- 2016/04/08 00:00 [revised]
PHST- 2016/05/13 00:00 [accepted]
PHST- 2016/05/29 06:00 [entrez]
PHST- 2016/05/29 06:00 [pubmed]
PHST- 2017/06/01 06:00 [medline]
AID - S0003-2697(16)30085-9 [pii]
AID - 10.1016/j.ab.2016.05.010 [doi]
PST - ppublish
SO  - Anal Biochem. 2016 Aug 15;507:21-6. doi: 10.1016/j.ab.2016.05.010. Epub 2016 May 
      24.