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Photon-enzyme interaction: The onset of a reactivation mechanism (

)

(1_{) Dipartimento di Biologia Animale, Università di Modena} Via Berengario 14, 4100 Modena, Italy

(2_{) Dipartimento di Fisica, Università di Milano - Via Celoria 16, 20133 Milano, Italy}

(3) Dipartimento di Fisica, Università di Modena - Via Campi 213-a, 41100 Modena, Italy (ricevuto il 5 Dicembre 1996; approvato il 4 Febbraio 1997)

Summary. — The cooperative photon-sustained reactivation of previously inhibited

ATPases is explained on the basis of a model where biochemical and physical aspects are conjugated. It is based upon the coupling between the electromagnetic field (connected to dipole excitations) and the chemical units responsible for the inhibition of the catalytic active centre, resulting into the site resumption of its normal active state, no photoelectric or thermal effects being required.

PACS 87.80 – Biophysical instrumentation and techniques.

Epidemiological studies on low-level exposure of humans to electromagnetic (em) radiation have become of public concern [1]. However, no consensus has been reached in the scientific community on the biological or pathological possible effects of these exposures [2]. On the other side, therapeutical applications are being established for an increasing number of different pathologies, within the broad range of the em spectrum, using for instance low frequency (with particular waveshapes) low intensity magnetic fields (typically in the range 40–80 Hz, and 1–3 mT) or alternatively low-energy, low-power lasers in the red and infrared portion of the spectrum (typically 1–30 mW and 500 to 900 nm). For pulsed magnetic fields let us recall the treating of fracture bones which would not unite and infected bones which would not heal, where patients have been spared with otherwise inevitable amputations [3, 4]. For the laser in the visible the pain relief and post-traumatic rehabilitation, especially in the sport medicine, have shown remarkable results in the absence of complementary therapies and of measurable thermal effects even of localized ones [5].

Biologist community seems little interested in biophysical theories that attempt to explain living systems from relatively general concepts based on “Quantum Biology”,

i.e. quantum collective interactions relying on the aspects of open dissipative systems

and of non-linear dynamics only, in conjunction with the fundamental

quantum-(†) In memory of Prof. Lorenzo Bolognani.

(*) Also at INFM (Istituto Nazionale di Fisica della Materia).

interactions, from an energetic point of view. The paradigm that impaired cell function equals impaired enzyme activity leads to the study of the inactivation and subsequent reactivation of ATPase enzymes. Atkinson [9] identified the “energy charge” (related to ATP concentration) as an overall parameter that keeps track of all the output and input activities with their energetic balance, so that it actually looks like an order parameter in the meaning of Haken approach to synergetics [10] that above all has the role of slaving all the related processes.

We will focus on those enzymes involved in muscular activity (myosin ATPase) whose energetic transduction is clear-cut to provide mechanical work. It is experimentally proved that chemical agents (CO2, 3 mol l21 urea) inhibit their activity

in vitro [11] providing effective inactivation agents. These in vitro results have their

counterpart in vivo in the muscular activity impaired by lactic acid. Furthermore it has been proven that those enzymes, which were previously inhibited can be reactivated (respect to controls) by a suitable low-energy low-power laser irradiation in the visible (HeNe laser, 10–20 mW, and exposure time between 10 to 40 min) without any detectable thermal effect [11]. Photomodulation of enzymes involving the inactivation and reactivation of enzyme reactions is being a field of interest in view of their widespread utility in biology and medicine [12]. A symmetric example is given by glycolysis which oscillates following circadian rythms. In glycolysis the phospho-fructokinase enzyme (PFK) [9] is the fundamental controller which possesses allosteric properties to maintain the energy balance of the cell, and therefore plays a role similar to that of an ATPase enzyme [13, 14].

The present work, conjugating the biochemical and physical points of view, provides the fundamentals for a model whose basic mechanism involves the interaction of photons with a previously impaired active centre (an ATPase enzyme). The proposed model, as far as the material point of view is concerned (i.e. all the molecular units belonging to the enzymatic reaction) is strongly related to the “concerned mechanism” of MWC [15] or to the more general model proposed by Eigen [16] showing that cooperative (allosteric) effects arise from elementary direct enzyme-enzyme interactions. The fundamental difference here relies on the role of “photons” that in our approach have the role of fundamental energy units (exactly in the same form as they do have in elementary quantum mechanics) that are able to keep track of the energy balance through all the intermediate states crossed by the enzymatic reaction. The above interaction explains how the photon-induced reactivation of the enzyme activity in vitro is obtained in agreement with experimental data [11]. The model may

Fig. 1. – Left-hand side: Simplified structure of the catalytic site of myosin ATPase enzyme in its normal configuration with atomic groups belonging to relevant amino acids. The central upper part (full line) shows the intake of the inhibiting factor CO2and the transition from LHS to RHS

picture. Right-hand side: simplified structure of the above site in its inhibited configuration as an effect of the intake of CO2molecules. The central lower part (dashed line) shows how the enzyme

reverts to its normal active configuration (transition from RHS to LHS) as an effect of the interaction with photons hn which free the enzymatic site by releasing the CO2molecules. give clues to interpret the above experimental results in vitro, while introducing the concepts and results of coherent quantum biology along the lines of Prigogine dissipative structures [17] tailored for biological systems, taking into account the coupling between the photon field and chemical units through quantum-mechanical coherent excitations.

Enzymes are mostly polypeptide chains with a molecular weight up to about 5 Q 105 amu similar to proteins. They start out as a linear chain of amino acids that fold, through electrical forces, into a given complex 3-dimensional structure characterized by a minimum energy and therefore stable state, even though they have an extremely large number of possible different configurations, which have aroused the interest of several physicists [18]. The enzyme activity, as a biochemical catalytic unit, occurs at the active centre (catalytic site), a kind of pocket embedded into the massive protein structure, where three or more amino acids, situated relatively far from one another along the polypetide chain, as an effect of the folding and twisting of the chain, turn out to be adjacent. In myosin ATPase, they are serine (Ser), glutamine (Gln), cysteine (Cys) and threonine (Thr) as shown on the left-hand side of fig. 1 where the catalytic site is depicted, in simplified form, as a gulf of the protein in its normal (folded) configuration opened upward (towards the substrate). Only few atomic groups are shown, they belong to the aminoacid residues and are those which are important to bind the ATP substrate into the active site and carry out the biocatalytic process. On the left-hand side of fig. 1 from left to right: OH` belongs to Ser, H2N`C1O to Gln,

SH` to Cys and a further OH` to Thr. In normal conditions the substrate ATP comes into contact with the catalytic site of the enzyme through a complex recognition effect. In the presence of Mg2 1 _{the ATP molecule unit is bound by induced fit to the above}

atomic groups, the enzyme transconforms and a series of intermediate reactions occur which eventually lead to the rapid cleavage of ATP by a mechanism of the type:

both occupied and transconformed, the ATP substrate cannot enter andOor undergo the cleavage reaction: so the enzyme becomes inactivated.

The effect of radiating a laser source onto the inactivated enzyme is depicted by the dotted lines connecting the right with the left hand side of fig. 1, where CO2 is shown

to exit and the reaction occurs in the reverse mode with the reactivation of the enzyme to its former normal state.

The above biochemical model can be integrated with a complementary physical model of the structure of enzymes (a-helix) and the signal transduction of dipole charge and phonon along these one-dimensional structures, transduction that has simultaneously the property of coherence and non-Ohmic charge transport [19].

It is interesting to notice that the catalytic site (CS) is relatively small in size compared to the full enzymatic macromolecule (EZ) to which it belongs. Looking at a single isolated enzyme, one may think that EZ acts as a heat bath for the CS also accounting for the hydrophobic nature of CS leading to a spatial configuration embedded into the protein folded 3-dimensional structure [18]. A collection of CSs may be looked at as a domain surrounded by the remaining part of all the enzymes. The CS is not a static structure, but a complex dynamic system correlated with other identical systems (into the cell or into the extract) in an organized network (domain or phase) following the concepts of Prigogine’s dissipative structures [17]. Here, not only ionic units, but also dipole structuresOpolar states (the most common occurrence in protein-like units [20]) oscillate like giant dipoles while remaining in a stable state far from equilibrium [21]. This introduces non-linear interactions within the above-described domain of CS subunits which is interconnected with CS-CS interact-ion-driven cooperative processes. In this case an entire domain of a macromolecular system reacts as a unit, which means that it transforms as a whole [22]: such a concept may be applied to the CS domain. According to the Froelich theory of coherent excitation in biological systems, containing essentially long-range non-linear features (typical of the present case), metastable highly polar states may arise where excitations may be supplemented by small conformational changes [23] and this applies in toto to the CSs upon their denaturationOimpairment. In such a case the specific modes of coherent excitation become quantum thermally decoupled from the remaining part of the system, where the EZ will act as a randomizing heat bath. Coherent excitation of a single mode of polar modes may arise at a rate S to all or only some of these modes, provided S exceeds a critical value S0 and provided these modes are in strong

in terms of non-linear optics and Quantum Field Theory [24] showing that the basic theory described by Haken [10] can be used. Among the fundamental results of this identification, there is the fact that the decay time of single active sites (i.e. the spontaneous decay time, or more clearly the survival rate of isolated excited states) decreases as a function of the cooperation degree of the elemental units. The cooperation degree can be monitored by the internal field, i.e. the em field of resonant type that is trapped inside the arrays of elemental units.

Let us now take a collection of enzymes and irradiate them with a laser source having the following two characteristics according with experimental data: i) low photon energy, so that non-ionisation occurs (typically in the red to near infrared range), and ii) low energy density, so that non-thermal effects can be foreseen in agreement with experimental evidence [11]. However, this radiant energy input is sufficient to raise S above S0providing a trigger action: “this gives a step-like response

similar to a phase transition” [23]. Consequently the final result is that the matrix element value for the photon interaction involving the transition BSKUS is highly enhanced inducing the photon-assisted reactivation of the enzymatic site, i.e. the de-inhibitionOreactivation of the enzymatic activity [11]. The transition from the inactivated state to the activated one is clearly the phase transition referred to by the Froehlich theory.

In conclusion a model suitable to explain the reactivation effect by power low-energy laser irradiation of previously inactivated enzymes is presented. It conjugates the biochemical data concerning the biological structures with a physical quantum-mechanical description of the biological system in terms of long-range forces in the framework of the general theory of non-linear dynamics of open, dissipative, far from equilibrium systems. No classic photoelectric effects are introduced at the level of ionic to covalent bond energies and, because of the quantum thermal decoupling, no thermal effects arise too, both in agreement with experimental data.

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