Kramers' Theory and the Dependence of Enzyme Dynamics on Trehalose
Total Page：16
File Type：pdf, Size：1020Kb
catalysts Review Kramers’ Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity José G. Sampedro 1,* , Miguel A. Rivera-Moran 1 and Salvador Uribe-Carvajal 2 1 Instituto de Física, Universidad Autónoma de San Luis Potosí, Manuel Nava 6, Zona Universitaria, San Luis Potosí C.P. 78290, Mexico; [email protected] 2 Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México C.P. 04510, Mexico; [email protected] * Correspondence: [email protected]fisica.uaslp.mx; Tel.: +52-(444)-8262-3200 (ext. 5715) Received: 29 April 2020; Accepted: 29 May 2020; Published: 11 June 2020 Abstract: The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics of proteins and water molecules in the bulk and the protein hydration shell. Enzyme catalysis and other processes dependent on protein dynamics are affected by the viscosity generated by trehalose, as described by the Kramers’ theory of rate reactions. Enzyme/protein stabilization by trehalose against thermal inactivation/unfolding is also explained by the viscosity mediated hindering of the thermally generated structural dynamics, as described by Kramers’ theory. The analysis of the relationship of viscosity–protein dynamics, and its effects on enzyme/protein function and other processes (thermal inactivation and unfolding/folding), is the focus of the present work regarding the disaccharide trehalose as the viscosity generating solute. Finally, trehalose is widely used (alone or in combination with other compounds) in the stabilization of enzymes in the laboratory and in biotechnological applications; hence, considering the effect of viscosity on catalysis and stability of enzymes may help to improve the results of trehalose in its diverse uses/applications. Keywords: trehalose; viscosity; enzymes; protein dynamics; Kramers’ theory; protein stabilization; enzyme inhibition 1. Trehalose in Biology The disaccharide trehalose has been used as food or therapeutic agent by humans since ancient times [1]. Trehalose is naturally found in the sweet cocoon (the capsule named trehala-manna) that protects the larvae of weevils, insects in the genus Larinus [1–4]. Pure trehalose crystals were isolated first from the ergot of rye by Wiggers (1832), then the carbohydrate was identified as the main component of trehala-manna (from which it takes the name trehalose) by Berthelot (1858) and identified as a non-reducing disaccharide formed by two glucose units [5]. Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside) was found at high concentrations in diverse organisms [6] and under stress conditions [4], including yeast and fungi [7,8], plants [9], and others [6]. Trehalose, in addition to forming part of cellular structures [6], has been claimed to behave as a reserve carbohydrate as it is usually found in spores of yeast, fungi, and bacteria [4,6–8,10], and also as a biostructure stabilizer, namely of proteins and membranes during harsh environmental conditions [4], and even in the pathogenesis of virulent bacteria (like actinomycetes and mycobacteria) and fungi [11,12]. Currently, the metabolic pathways for trehalose biosynthesis and hydrolysis, and their regulation are well known [8–10,13,14], and in some organisms, trehalose metabolic pathways seem to have a major role in the regulation of other metabolic processes [7,8]. Catalysts 2020, 10, 659; doi:10.3390/catal10060659 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 659 2 of 19 Among the diverse stress conditions where trehalose is synthesized and accumulated, dehydration (or the phenomenon of anhydrobiosis) has been that which has attracted the most interest (because of its potential industrial applications) [5,15–17], namely in food and pharmaceutics. Van Leeuwenhoek (1702) first described the phenomenon in bdelloid rotifers [18,19]. Since then, other organisms have been recognized to endure the absence of water, such as nematodes, brine shrimp cysts, yeast, and some plants [4,6,18]. Experiments have demonstrated the effectiveness of trehalose to preserve the structure of proteins and membranes in the dry state [16,20,21]. Nonetheless, the role of intrinsically disordered proteins (stress proteins) protecting organisms that synthesize little to no trehalose seems to be important [22,23]. In nature, the dehydration process in anhydrobiotic organisms seems to be slow, i.e., water is removed in minute amounts leading to cell desiccation [16]. At the same time, trehalose begins to be synthesized, reaching high concentrations in the cytoplasm, while a small amount is exported (trehalose is required at both surfaces of the plasma membrane) [24]. Although cell survival does correlate with the amount of trehalose present in the cell [16,19], other biochemical responses contributing to anhydrobiosis (or acting synergically with trehalose) seem to take place simultaneously to trehalose accumulation [16], e.g., the synthesis of heat shock proteins [25]. Physically, under optimum growth conditions (nutrients, temperature, etc.) the cell cytoplasm is a crowded space containing a great diversity of molecules, hence inherently highly viscous [26–29]. Then, during dehydration, the accumulation of trehalose increases viscosity even further as water-loss proceeds [30–34]. Hence, viscosity increases up to finally reach the glassy state [34,35]. Here, although the interaction of trehalose with proteins has been subjected to some controversy [36], the effect of trehalose on the maintenance of the integrity of membranes (via the interaction with phospholipid headgroups) is relatively well understood [16,37]. Nonetheless, what is certain is that viscosity (the glassy state) generated by trehalose is high. Here proteins and other bio-structures are “arrested”, which in turn preserves both the structure and function of proteins [34,38–40]. In microorganisms, trehalose is also synthesized in the stationary phase of growth. In yeast cultures when nutrients are exhausted, cells enter a quiescent state known as the stationary phase [41], where the metabolic rate is slowed and cell division stops (arrested cell cycle) [42,43]. Notably, during entry to the stationary phase, trehalose is accumulated at high concentrations (up to 20% in dry weight), as well as glycogen (up to 8% in dry weight) [42]. The cells at the stationary phase thus become denser, heavier, and cytoplasm viscosity increases [31,32,42]. In this regard, trehalose seems to have a role in the exit of yeast from the stationary phase besides [42]; however, no specific role of trehalose in cell metabolism under the quiescent state has been described. Instead, cells in the stationary phase display high thermotolerance, possibly due to trehalose accumulation [4,21]. 2. Trehalose, Water, and Proteins Trehalose/water interaction is the basis for protein stabilization by trehalose [36,44–46]. Water is the medium where most biochemical processes take place and the hydration water has a fundamental role in protein structure and function [47–49]. In this regard, water molecules seem to be more uniformly distributed on the surface of globular proteins when compared to those on intrinsically disordered proteins (IDPs) [48,50]; despite that, the arrangement of tetrahedral water molecules is more disordered on the surface of globular proteins than on IDPs [50]. The above is probably due to the highly irregular surface of globular proteins. Interestingly, the dynamics of hydration water on proteins are different from bulk water molecules [47,48,51–53]. In solution, trehalose molecules display a relatively rigid conformation and slow translational motion (self-diffusion) [46,54]. Besides, trehalose molecules interact through trehalose–trehalose hydrogen bonds [44,55,56]; while trehalose–water hydrogen bonds are weaker [56]. This seems to facilitate vitrification [46], i.e., by allowing water molecules to escape from the system. Additionally, trehalose appears to be physically (preferentially) excluded from protein surfaces [57,58], thus promoting preferential hydration [45,59–61]. This is while maintaining the disordered arrangement of hydration water molecules [50]. Nonetheless, the dynamics Catalysts 20202020,, 10,, 659x FOR PEER REVIEW 3 of 19 Nonetheless, the dynamics of hydration water appears to be slowed down by trehalose and consequently,of hydration water dynamics appears of tothe be amino slowed acid down residues by trehalose in proteins and consequently, decrease [45,48,61 dynamics–67] of, e.g., the aminoin C- phycocyaninacid residues in(CP), proteins trehalose decrease slows [45 ,48down,61– 67the], e.g.,dynamics in C-phycocyanin of the hydration (CP), trehalose water by slows an downorder theof magnitudedynamics of when the hydration compared water to the by dynamics an order of of bulk magnitude water [68]. when As compared a result, protein to the dynamics processes ofwhere bulk waterstructural [68]. dynamics As a result, are protein a major processes component where become structural affected dynamics by the are presence a major componentof trehalose become in the medium,affected by namely the presence folding, of unfolding, trehalose inthermal the medium, and cold namely inactivation, folding, oligomerization, unfolding, thermal aggregation, and cold catalysisinactivation,, and oligomerization, even the