Effects of Pressure on the Kinetics of Capture by Yeast Alcohol Dehydrogenase

Sato

et al. 2008

Biotechnology Progress

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD + ) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine). The YADH-and NAD + -containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD + concentrations were 2.3 mg/ mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD + was examined on the thermal stability of YADH at 45 and 50°C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD + as well as the free YADH with and without NAD + . Free YADH was increasingly deactivated during its incubation at 45°C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD + at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD + was lowered at 50°C. The turbidity measurements for the above free YADH solution with and without NAD + revealed that the change in the enzyme tertiary structure was much more pronounced at 50°C than at 45°C even in the presence of NAD + . This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD + . On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50°C much higher than those of the above free enzyme systems, YADH/NAD + and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD + at 50°C than at 45°C. This was considered to be because even at 50°C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD + in liposomes.

Section: Resultssupporting

confidence: 52%

Liposomal Encapsulation of Yeast Alcohol Dehydrogenase with Cofactor for Stabilization of the Enzyme Structure and Activity

Sato

et al. 2008

Biotechnology Progress

“…83,84 Transient kinetic evidence is lacking for an enzyme–coenzyme isomerization for the yeast enzyme, but effects of pressure on V / K for benzyl alcohol oxidation led to the suggestion that the E–NAD + ↔ E*–NAD + equilibrium constant is 75 ± 13. 85 Hydrogen–deuterium exchange studies also suggest that binding of NAD + results in a conformational change. 86 …”

Section: Resultsmentioning

confidence: 99%

Yeast Alcohol Dehydrogenase Structure and Catalysis

2014

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure.

“…This is a challenging task using conventional high‐pressure cells since loading and pressurising the reaction cell before measurement can commence takes considerable time and requires the use of low enzyme concentrations 21. 22 To overcome these limitations, we have used an alternative method that employs a high‐pressure rapid‐mixing stopped‐flow (HPSF) instrument. Using a HPSF, we initiate the reaction in situ by mixing, for example, a pre‐equilibrated solution of DHFR and NADPH with an equal volume of H 2 F. Representative reaction traces are shown in Figure S3 in the Supporting Information.…”

Section: Resultsmentioning

confidence: 99%

Are the Catalytic Properties of Enzymes from Piezophilic Organisms Pressure Adapted?

et al. 2009

We report the crystal structure of dihydrofolate reductase (DHFR) from the psychropiezophilic bacterium Moritella profunda, which was isolated from the deep ocean at 2 degrees C and 280 bar. The structure is typical of a chromosomal DHFR and we were unable to identify any obvious structural features that would suggest pressure adaptation. In particular, the core regions of the enzyme are virtually identical to those of the DHFR from the mesophile Escherichia coli. The steady-state rate at pH 9, which is limited by hydride transfer at atmospheric pressure, is roughly constant between 1 and 750 bar, falling at higher pressures. However, the value of K(M) increases with increasing pressure, and as a result k(cat)/K(M) decreases over the entire pressure range studied. Isotope effect studies showed that increasing the pressure causes a change in the rate-limiting step of the reaction. We therefore see no evidence of pressure adaptation in either the structure or the activity of this enzyme.