Biomimicry Enhances Sequential Reactions of Tethered Glycolytic Enzymes, TPI and GAPDHS

<div><p>Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.</p></div

Properties of recombinant proteins.

<p>Enzyme activities in solution were determined as described. K_m values are for the substrates, glyceraldehyde 3-phosphate (GAP) for TPI and 3-phosphoglyceric acid (3-PGA) for GAPDHS.</p

Site-specific immobilization improved the coupled reaction of tethered enzymes.

<p>a) Forward TPI-GAPDHS coupled reaction. b) Reverse GAPDHS-TPI coupled reaction. Both enzymes were tethered via their His tags and site-specific immobilization (); both enzymes were tethered via carboxyl-amine binding (); control chips had no attached protein, but the complete reaction mixtures () [n = 9 (a), n = 12 (b); mean values are plotted with SE]. c) Comparison of forward TPI-GAPDHS activities at the 50 min timepoint calculated from (a). d) Comparison of reverse GAPDHS-TPI activities at the 60 min timepoint calculated from (b). Site-specific immobilization of His-NiNTA showed significantly higher activity (*p = 0.0001 ** p<0.001).</p

Design of recombinant proteins and verification of purified His-TPI and His-GAPDHS.

<p>a) A hexahistidine tag was introduced to modify the amino terminal, germ cell-specific domain (gcs) of sperm TPI. b) SDS-PAGE showing representative coomassie brilliant blue (CBB) protein staining and immunoblot analysis of purified His-TPI with antibodies against the His-tag (His) and the protein (TPI). c) A hexahistidine tag replaced the amino-terminal proline-rich domain (PRD) of GAPDHS. d) SDS-PAGE showing representative protein staining (CBB) and immunoblot analysis of purified His-GAPDHS with antibodies against the His-tag (His) and the protein (GAPDHS).</p

Site-specific immobilization improved specific activities of tethered enzymes.

<p>For both TPI and GAPDHS, site-specific immobilization using the His tag significantly improved enzyme specific activities versus carboxyl-amine binding. Although the total amounts of TPI (a) and GAPDHS (c) immobilized to carboxyl (C-A) or Ni-NTA activated surfaces were statistically identical, the specific activity of His-TPI bound to Ni-NTA was significantly higher than when bound via carboxyl-amine attachments (b; *p = 0.0143, n = 9). Similarly, the specific activity of His-GAPDHS was higher when bound to Ni-NTA versus carboxyl-amine binding (d; **p = 0.0234, n = 7).</p