Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.
Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
James P
JP
Lata
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Lizeng
L
Gao
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Chinatsu
C
Mukai
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Roy
R
Cohen
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Jacquelyn L
JL
Nelson
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Lynne
L
Anguish
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Scott
S
Coonrod
Baker Institute for Animal Health, ‡Biomedical Engineering, and §Atkinson Center for a Sustainable Future, Cornell University , , Ithaca, New York 14853, United States.
Alexander J
AJ
Travis
D000327:Adsorption; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D053768:Metal Nanoparticles / Q000737:chemistry*; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010316:Particle Size; D011487:Protein Conformation
D004800:Enzymes, Immobilized; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D006639:Histidine; D006046:Gold