The enzyme alcohol dehydrogenase (ADH) of yeast is one of the interesting objects in the chemical, biological, pharmaceutical, electrochemical, agrochemical and aromatic industries [1,2,3,4,5]. For example, in [5], a successful preparation of hexanal catalyzed by ADH from baker’s yeast was carried out. Essential to the functionality of ADH is that it must be added in stoichiometric amounts and cannot be replaced by more economical synthetic products. At the same time, the coenzyme NAD+ (nicotinamide adenine dinucleotide) is widely used in biocatalytic oxidations catalyzed by ADH. The most intriguing in the structural aspect of ADH, as shown by X-ray crystallography, is that ADH undergoes global conformational changes (it does not matter when binding the NAD+—oxidized form or the NADH—reduced form of NAD), including rotation of the catalytic domain relative to the coenzyme binding domain and rearrangement of the active center to form a catalytically active enzyme. A change in conformation requires the presence of a complete coenzyme and depends on various chemical or mutational substitutions. These substitutions can enhance catalytic activity by altering the kinetics of isomerization and the rate of coenzyme dissociation [2,3,4,5,6].

As for the structural aspect of the enzyme, the orientation of ADH on various sorbents and electrically conductive matrices was studied using experimental observations and mathematical modeling of the protein dependence on the pH of the solution. As is known, the deactivation of the enzyme is caused by unsuitable conditions (temperature, pH), which leads to a change in the activity of the enzyme ADH. In relation to the problems of modeling the effect of pH on the orientation of protein sorption, the sorption behavior of the ADH enzyme based on specific interactions of protein groups with groups on the surface and its charge was previously studied in [7,8,9,10,11,12,13,14,15]. It was assumed that the conformation of the ADH enzyme corresponds to its conformation in the crystal structure and does not change when the surrounding solution and the surface of the sorbent change, which is a rather rough approximation. The implementation of various options includes, for example, the immobilization of a two-substrate enzyme on the surface of electrode materials [7,8,9,10,11,12,13,14,15].

However, it should be noted that the experimental study of the above issues is difficult. Therefore, in recent years, computational and simulation analysis methods have been widely used for these purposes [16,17,18,19,20,21,22,23]. This study utilized computer molecular dynamics (MD) modeling to study the structural conformational changes of the enzyme ADH and its cofactor NAD, which occur in aqueous solution when interacting with a graphite carbon surface. The MD analysis data provide a significant extension of the initial basic model, thereby allowing the protein conformation to be changed in the region of titrated amino acid residues of ADH in atomic/molecular details. The identification of the characteristic conformation of key titrated amino acids may become a necessary stage for further research and the implementation of a numerical experiment, which will be carried out by varying the pH and surface charge values [2,7,8,9,10,11,12,13,14,15].

Of course, the current research correlates with several parallel theoretical and experimental works concerning the immobilization and adsorption of enzymes on a carbon surface, the location and fixation of enzymes on carbon platforms, as well as on the surfaces of bioanodes, etc. It is worth noting that the choice of carbon surfaces and platforms is due to good control of the location of enzymes on the surface with very low enzyme consumption. To date, the kinetic behavior of the ADH enzyme in a solution fixed on carbon platforms has been studied. However, there are few comparative studies of the ADH enzyme that take into account the kinetic behavior of the enzyme, despite the studies of the structure and kinetic mechanism of immobilized enzymes available in the literature. Kinetic parameters obtained for the ADH enzyme attached to a carbon surface (carbon fiber paper) indicate a decrease in activity after the immobilization and fixation process. A significant loss of enzymatic activity was observed after immobilization, despite the retention of the enzyme’s affinity for its substrates and coenzymes. It is believed that MD observations of the structural behavior of ADH+NAD, diffusion and adsorption processes correlate well with the kinetic parameters obtained for the enzyme fixed with ADH on a carbon platform. Various kinetic parameters (mainly the concentration of substrates and coenzyme) indicate a change (loss or decrease) in enzymatic activity of the immobilized enzymes following the surface immobilization process. In particular, the observations obtained in the current study may correlate well with practical applications, for example, with the growing interest of biotechnologists in the use of immobilized enzymes such as ADH for many purposes, bioremediation, sensors and biofuel elements, etc.

Figure 2 shows the resulting pictures of the adsorption of ADH+NAD on the surface of graphite carbon during prolonged 100 ns of dynamic changes from (a) the initially relaxed state to (b,c) intermediate states and (d) the final equilibrium condition.

The enzyme ADH+NAD undergo multimillion conformational and rotational changes before adsorption on this graphite carbon (C-surface) in order to finally capture and relax on the surface. The dynamics of ADH+NAD adsorption on the graphite carbon surface was monitored using MD/AMBER calculations and Visual Molecular Dynamics (VMD) software. For each MD model, calculations were performed in 100 ns using the module “pmemd.cuda”. One of the nontrivial events of the conformational structural dynamics of the entire ADH+NAD+water system/C-surface and tracking of individual amino acid residues should be the behavior of the catalytic loops of the enzyme. Figure 2 show the dynamic patterns of the ADH+NAD/C-surface and the adsorption processes, accompanied by a gradual change in the orientation of the two catalytic loops of the ADH+NAD molecule relative to the graphite surface. An important observation is that these two catalytic loops are located close together inside the ADH+NAD molecule. However, upon reaching the adsorbing graphite surface, a separation between these loops is observed. A visual description of the entire process has shown in Figure 2, where two enlarged loops separate from each other and, consequently, open important catalytic ADH fragments as a result of the influence of the adsorbing C-surface of these two enzyme loops.

Further, Figure 3 show the location of the above-mentioned two catalytic loops of the ADH+NAD molecule relative to the graphite surface. Two terminal amino acid residues THR290 and ILE317 are highlighted here, where Δ is introduced, as the distance between these two terminal amino acids.

Figure 3a shows the location of the catalytic loops during adsorption of the ADH+NAD molecule on the carbon surface of graphite in the final (100 ns) state. Figure 3b shows the results of calculations of the dynamics of the distance between the catalytic loops of ADH+NAD on the carbon surface of graphite as a function of time. The results in Figure 3 clearly confirm the important observation mentioned above that the two catalytic loops separate from each other when they reach the adsorbing surface of graphite, whereas initially they were located close to each other inside the ADH+NAD molecule.

The above observation and the presented data on the dynamics of the catalytic loops of ADH+NAD on the carbon surface correlate with the dynamical changes and rotations of the coenzyme NAD inside the ADH molecule. Using the data in Figure 3, the conformational changes of NAD during adsorption of ADH on the surface were also estimated, along with the open gap between the catalytic loops. In Figure 4, the positions of the atoms at the beginning and end of the chain NAD, as well as the atoms in the central region of NAD, are noted.

Figure 4 demonstrate a method for estimating the angle of γ-vibration and rotation of coal in the three-dimensional structure of the NAD molecule, where auxiliary vectors are introduced and the starting points are designated A~ (PA, O3, PN), B~N_begin, C~N_end with the results of calculating the dynamical changes in the angle of γ of the NAD molecule depending on time. They are shown in Figure 4b below. It can be seen that the dynamical changes of γ with time are completely correlated with the dynamics of the change in the distance between the catalytic loops of ADH + NAD on the carbon surface of graphite. The comparison of the results in Figure 3 and Figure 4 is straightforward. Thus, these results are the key features of the entire process of adsorption of ADH+NAD/C-surface, which is accompanied by a non-trivial structural transformation of the coenzyme NAD, correlating with the behavior of the catalytic loops of the enzyme ADH.

Further, below in Figure 5, the orientations of the three titrated amino acid residues ASP54, HIS252 and ASP268 with time are shown.

It is worth noting that for ADH+NAD+water+graphite surface, as described above, all calculations were performed at pH = 7 in a aqueous medium. The experimental data and analysis of the kinetic behavior of the ADH enzyme in solution as a function of pH and temperature, have shown that the highest activity is achieved in the pH range 7.0–8.0 and an optimal temperature about 303 K [4,5,6,7,26,27,28]. At pH 7, the protonation states of the catalytic triad, histidine (HIS), aspartate (ASP), and glutamate (GLU), are determined by their respective pKa values. Histidine has an imidazole side chain with a pKa of approximately 6.0, meaning that at physiological pH, it exists primarily in its deprotonated, neutral form. Aspartate (ASP) and glutamate (GLU), with pKa values of around 3.9 and 4.2, respectively, are both fully deprotonated at pH 7.

In Figure 6, the dynamics of the angle of γ—internal vibration and rotation (N-CA-OD2) of three titrated amino acid residues ASP54 (a), HIS252 (b) and ASP268 (c) have been calculated. As in the case of the molecule above, auxiliary vectors were introduced, thereby thus, choosing the places of evaluation of amino acid residues in the form of: A~(CA)—the middle atom of the residue; B~N_begin (NH3-end)—the residue of one terminal atom; C~OD2 (NE2)—the residue of another terminal atom. From the results shown in Figure 6, it can be seen that the dynamic behavior and the change in γ over time fully correlate with the dynamics of the change in the distance between the catalytic loops of ADH + NAD on the carbon surface of graphite, as shown in Figure 3b, as well as in Figure 4b.

Figure 7 shows the location of the catalytic loops during adsorption of the ADH+NAD molecule on the carbon surface of graphite in the initial (a) state, (b) in the intermediate (50 ns) and (c) in the final (100 ns) states.

Figure 8 demonstrates the procedure for calculating the estimate of the angle of γ-vibration and rotation of coal in the three-dimensional structure of the molecule NAD, where auxiliary vectors are introduced and the starting points are designated A~(PA,O3,PN), B~N_begin, C~N_end. The dynamical changes in the angle γ of the NAD molecule as a function of time correlate with the dynamics of changes in the distance between the catalytic loops of ADH+NAD on the carbon surface of graphite:

In Figure 8 below, the calculation of the dynamics of the NAD structure over time includes the angle between (point A~(PA, O3, PN), point B~N_begin, point C~N_end).

Thus, the orientation characteristics of the ADH enzyme’s titratable amino acid residues strongly correlate with the transformation of the NAD coenzyme. Thereby, the complete ADH+NAD structural behavior has to occur on the graphite surface during the adsorption processes. The ambient temperature and water environment during the implementation of the MD numerical experiment has been chosen. So far, the results in terms of pH and temperature coincide with data reported in the literature for the ADH enzyme in solution. Perhaps the key results, particularly Figure 3b and Figure 4b, which represent the main discoveries or observations, correlate well with experiments on the kinetics of the ADH enzyme. As can be seen from the MD results, presented in Figure 3b and Figure 4b, during the ADH+NAD adsorption the diffusion processes occur for 55 ns; after this period, ADH+NAD touch the graphite surface for a final relaxation on the surface. Following the adsorption of the ADH enzyme on the carbon surface, the coenzyme NAD from 55–60 ns begins to fluctuate, which is in full correlation with the opening of two catalytic loops associated with the NAD region. It is worth noting that current observation correlates with some experimental work related to the immobilization and adsorption of ADH enzyme on the carbon surface, the location of the enzyme and its fixation on carbon platforms. Moreover, the results also show that, in order to maintain an environment in which the ADH enzyme display good activity and to provide conditions for future technological applications, physiological conditions and ambient temperature can satisfactorily be applied to an enzymatic system involving the dehydrogenase enzyme. At the same time, the choice of carbon surfaces and platforms is motivated by a good control of the location of enzymes on the surface with very low enzyme consumption. The kinetic rates obtained for the ADH enzyme fixed to the carbon surface indicates a decrease in activity after the immobilization and fixation process. A significant loss of enzymatic activity is observed after immobilization, even though the affinity between the enzymes and their substrates and coenzymes persists. To date, the MD observations of the structural behavior of ADH+NAD, diffusion and adsorption processes correlate well with the kinetic rates obtained for an enzyme anchored by ADH on a carbon platform with various kinetic parameters (mainly the concentration of substrates and coenzyme), indicating the substantial changes (the loss or decrease) in the enzymatic activity of fixed enzymes after the immobilization process on the carbon surface. Judging by experiments and kinetic studies, this is due to a significant loss of enzymatic activity of ADH in the presence of a carbon surface, and the anchored enzyme should lose enzymatic activity. Thus, for the ADH enzyme and NAD coenzyme, the carbon platform has probably cause not only diffusion activity, but also some restriction in diffusion process, thereby making them difficult for the reduced form of the coenzyme to escape from the bulk solution (see, Supplementary Materials).

In conclusion, the structural conformations of the alcohol dehydrogenase (ADH) enzyme with its cofactor nicotinamide adenine dinucleotide (NAD) were studied on a graphite-like surface (C-surface) using the MD (molecular dynamics) modeling method. The enzyme ADH+NAD was modeled in an aqueous medium. The MD simulations, conducted using the AMBER-18 package (a fast implementation of the pmemd.cuda module), provide valuable statistical data on key structural aspects of ADH, particularly its conformational changes, including the rotation of the catalytic domain, the coenzyme binding domain, and rearrangements of the active center. This analysis is essential for understanding the atomic and molecular details of a catalytically active enzyme. The 100-ns dynamic simulations allowed to track the conformational and rotational changes of ADH+NAD in an aqueous environment.

A non-trivial phenomenon has been observed: the conformational changes in ADH were correlated with structural transformations in its cofactor, NAD. However, it remains unclear whether the catalytic activity of ADH is preserved after such extensive conformational changes. Furthermore, the findings align with experimental studies on enzyme immobilization and adsorption onto carbon surfaces, as well as enzyme fixation on carbon platforms and oxide surfaces.

The MD results of the distance and angle with time, appear to correlate well with experimental data on ADH+NAD kinetics, where the ADH+NAD diffusion process occurs over approximately 50–60 ns, after which the complex reaches the graphite surface and undergoes final relaxation. Following ADH adsorption onto the carbon surface, NAD begins to fluctuate between 55 and 60 ns, aligning with the opening of two catalytic loops associated with the NAD-binding region. Experimental and kinetic studies suggest that this structural change significantly impacts enzymatic activity, leading to the loss of catalytic function in the immobilized enzyme. For ADH and NAD species on carbon platforms, diffusion restrictions may arise, potentially preventing the reduced form of the coenzyme from flowing freely in the bulk solution. The ADH+NAD complex was preserved in the simulations, with the cofactor modeled within the enzyme structure as in the original PDB file. Interestingly, NAD can be efficiently stabilized outside the enzyme, maintaining its catalytic efficiency in a selected NAD-enriched solution, a specially designed buffer or solvent system to stabilize and preserve its activity.