Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes

Abstract

We studied the combined effects of pressure (0.1-200 MPa) and temperature (22, 30, and 38 8 8C) on the catalytic activity of designed amyloid fibrils using ah igh-pressure stopped-flows ystem with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed ar emarkably high pressure and temperature stability of the fibrillar systems.H igh pressure enhances the esterase activity as ac onsequence of an egative activation volume at all temperatures (about À14 cm 3 mol À1 ). The enhancement is sustained in the whole temperature range covered, which allows af urther acceleration of the enzymatic activity at high temperatures (activation energy 45-60 kJ mol À1 ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.Amyloid fibrils are linear polypeptide aggregates with ah ighly periodic cross-b sheet conformation, which abnormally occur inside the body and are associated with diseases such as Alzheimersa nd Parkinsons. [1] Increasing evidence shows,h owever,t hat amyloid structures may also occur as functional biological units in vivo,a nd there is considerable interest in the material sciences and bionanotechnology to exploit the unique structural features of these highly stable, nanostructured, and biodegradable assemblies for the generation of novel applications and technical tools,s uch as protein-based catalysis,conducting nanowires,and devices for water purification. [2] Ak ey factor limiting the further utility of amyloid materials and their incorporation into technical devices is their so far unclear stability and performance under processrelevant conditions.T oe stablish these characteristics we herein describe the results obtained by exposing two recently described preparations of de novo designed and catalytically active fibril systems to different types of environmental stress. These two systems consist of the peptides Ac-LHLHLRL-CONH 2 (AF1) and Ac-IHIHIQI-CONH 2 (AF2) that form amyloid fibrils in vitro,w hich exhibit, upon Zn 2+ binding,a n esterase activity that can be spectroscopically followed by the hydrolysis of the substrate p-nitrophenyl acetate (pNPA). [3] Tr ansmission electron microscopy (TEM) showed that the fibril morphology of both samples is markedly different. Both samples contain polymorphic fibrils,a sc an be inferred from the broad variations of the fibril width (Figure 1), and there are considerable variations in the quaternary structure of the fibrils in these samples.A F1 fibrils are relatively long and twisted ribbons ( Figure 1A), while AF2 fibrils exhibit amore plate-like morphology ( Figure 1B).We then determined the effect of different environmental conditions,s uch as high hydrostatic pressure (HHP) and temperature,o nt he structural stability and catalytic activity of the two fibril enzymes.U ntil now the effects of HHP on enzymatic substrate conversions have been characterized only sporadically. [4] HHP would be expected to accelerate enzymatic reactions,i f...

Catalytic peptide assemblies

“…Interestingly, the K M was practically not affected by the pressure increase, whereas both K M and k cat were affected by temperature change. 137…”

High pressures increase α-chymotrypsin enzyme activity under perchlorate stress

“…Enzyme activity was measured using a high-pressure stopped-flow system, HPSF-56, from Hi-Tech Scientific 16 , 19 , 45 . The enzyme was prepared as a 20 µM buffered stock solution (0.1 M Tris- HCl, 0.01 M CaCl 2 , pH 7.8) in the absence or presence of MgSO 4 or Mg(ClO 4 ) 2 at either 0.25 or 0.5 M. Enzyme concentration was measured using its absorbance at 280 nm with a molar extinction coefficient of 51000 M −1 cm −1 .…”

Vortex Fluidic Chemical Transformations

“…This approach could allow a large range of enzymes to be accelerated at standard temperature and pressure with little optimization, and importantly, no genetic modification. Currently, high-pressure reactors can drive enzymatic transformations, [31] it is thus conceivable to believe that a room temperature and pressure approach could be beneficial.…”