PMID- 22148353
OWN - NLM
STAT- MEDLINE
DCOM- 20120515
LR  - 20240323
IS  - 1520-6882 (Electronic)
IS  - 0003-2700 (Print)
IS  - 0003-2700 (Linking)
VI  - 84
IP  - 2
DP  - 2012 Jan 17
TI  - Employing the metabolic "branch point effect" to generate an all-or-none, 
      digital-like response in enzymatic outputs and enzyme-based sensors.
PG  - 1076-82
LID - 10.1021/ac202701c [doi]
AB  - Here, we demonstrate a strategy to convert the graded Michaelis-Menten response 
      typical of unregulated enzymes into a sharp, effectively all-or-none response. We 
      do so using an approach analogous to the "branch point effect", a mechanism 
      observed in naturally occurring metabolic networks in which two or more enzymes 
      compete for the same substrate. As a model system, we used the enzymatic reaction 
      of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction 
      catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low 
      substrate concentrations, the second enzyme outcompetes the first, turning off 
      the latter's response. Above an arbitrarily selected "threshold" substrate 
      concentration, the nonsignaling HK enzyme saturates leading to a "sudden" 
      activation of the first signaling GOx enzyme and a far steeper dose-response 
      curve than that observed for simple Michaelis-Menten kinetics. Using the 
      well-known GOx-based amperometric glucose sensor to validate our strategy, we 
      have steepen the normally graded response of this enzymatic sensor into a 
      discrete yes/no output similar to that of a multimeric cooperative enzyme with a 
      Hill coefficient above 13. We have also shown that, by controlling the HK 
      reaction we can precisely tune the threshold target concentration at which we 
      observe the enzyme output. Finally, we demonstrate the utility of this strategy 
      for achieving effective noise attenuation in enzyme logic gates. In addition to 
      supporting the development of biosensors with digital-like output, we envisage 
      that the use of all-or-none enzymatic responses will also improve our ability to 
      engineer efficient enzyme-based catalysis reactions in synthetic biology 
      applications.
FAU - Perez Rafael, Sandra
AU  - Perez Rafael S
AD  - Sensors and Biosensors Group, Department of Chemistry, Autonomous University of 
      Barcelona, 08193 Bellaterra, Catalonia, Spain.
FAU - Vallee-Belisle, Alexis
AU  - Vallee-Belisle A
FAU - Fabregas, Esteve
AU  - Fabregas E
FAU - Plaxco, Kevin
AU  - Plaxco K
FAU - Palleschi, Giuseppe
AU  - Palleschi G
FAU - Ricci, Francesco
AU  - Ricci F
LA  - eng
GR  - R01 AI076899/AI/NIAID NIH HHS/United States
GR  - R01 AI076899-01A1/AI/NIAID NIH HHS/United States
GR  - AI076899/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20111228
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
RN  - 0 (Enzymes, Immobilized)
RN  - EC 1.1.3.4 (Glucose Oxidase)
RN  - EC 2.7.1.1 (Hexokinase)
RN  - IY9XDZ35W2 (Glucose)
SB  - IM
MH  - *Biosensing Techniques
MH  - Catalysis
MH  - Electrodes
MH  - Enzymes, Immobilized/*metabolism
MH  - Glucose/*analysis/metabolism
MH  - Glucose Oxidase/*metabolism
MH  - Hexokinase/*metabolism
MH  - Humans
MH  - Kinetics
PMC - PMC3893712
MID - NIHMS346826
EDAT- 2011/12/14 06:00
MHDA- 2012/05/16 06:00
PMCR- 2014/01/16
CRDT- 2011/12/14 06:00
PHST- 2011/12/14 06:00 [entrez]
PHST- 2011/12/14 06:00 [pubmed]
PHST- 2012/05/16 06:00 [medline]
PHST- 2014/01/16 00:00 [pmc-release]
AID - 10.1021/ac202701c [doi]
PST - ppublish
SO  - Anal Chem. 2012 Jan 17;84(2):1076-82. doi: 10.1021/ac202701c. Epub 2011 Dec 28.