Membrane-bound enzymes coupling factor

The question of whether an enzyme is membrane bound or membrane associated is to some extent a matter of semantics. However, it is certainly true that some proteins are readily dissociated from membranes whereas others require quite drastic conditions before they can be dissociated from the membrane. As limiting cases, the former can be designated as membrane associated and the latter as membrane bound. Enzymes that are generally considered membrane bound are firmly embedded in the membrane structure. For example, the mitochondrial coupling factor is strongly coupled to the bilayer structure by hydrophobic polypeptides. The Na+-K+ ATPases that have been purified have a small patch of associated phospholipids when the enzyme is delipidated, enzymatic activity is lost. In fact, membrane-bound enzymes appear to be... 

The conformation of membrane-bound enzymes is undoubtedly restricted by the membrane. However, the mechanism of action of these enzymes appears to be similar to that of soluble enzymes, so that the presence of clefts and conformational flexibility is to be expected. The mitochondrial coupling factor apparently contains both the ATP synthesizing enzyme and a proton channel conformational changes undoubtedly play a role in the function of this system. A large movement of polypeptide chains has been proposed in the functioning of this system (and for other membrane-bound enzymes), but no convincing experimental evidence is available to support such a hypothesis. 

Fig. 11. Proposed function of electrochemical and Na potentials in energy conservation coupled to acetate fermentation to CH4 and CO2. The Na /H antiporter is involved in the generation of A/iH from A/iNa. CH3CO-S-C0A, acetyl-coenzyme A [CO], CO bound to carbon monoxide dehydrogenase CH3-H4MPT, methyl-tetrahydromethanopterin CH3-S-C0M, methyl-coenzyme M. The hatched boxes indicate membrane-bound electron transport chains or membrane-bound methyl-transferase catalyzing either IT or Na translocation (see Figs. 5, 6 and 12). It is assumed that enzyme-bound [CO] is energetically equal to free CO. ATP is synthesized via membrane-bound H -translocating ATP synthase. The stoichiometries of translocation were taken from refs. [107,234] n, X, y and z are unknown stoichiometric factors.

Laval etal. (1984) bound LDH covalently to electrochemically pretreated carbon. The enzyme was fixed by carbodiimide coupling simultaneously with anodic oxidation of the electrode surface. The total amount of immobilized LDH was determined fluorimetrically after removal from the electrode and hydrolysis. The authors found that at a maximal enzyme loading of 13 pmol/cm2 six enzyme layers are formed. The immobilization yield was about 15%. The kinetic constants, pmax and. Km, were not affected by the immobilization. The obtained enzyme loading factor of 10-3 indicates that diffusion in the enzyme layer was of minor influence on the response of the sensor. The layer behaved like a kinetically controlled enzyme membrane, i.e., the NADH oxidation current was proportional to the substrate concentration only far below Km- With increasing enzyme loading the sensitivity for NADH decreased due to masking of the electrode surface. 

In a precedent work (7) we have applied it to the fixation of ADP and ATP on chloroplast coupling factor CFl and on some of its subunits. This protein is a part of a proton translocating multisubunit enzyme found in photosynthetic membranes and is able to hydrolyse and synthesize ATP. We have shown by this method that CFl possesses six binding sites two high affinity sites for ADP or ATP (Kd=1-5 10 in addition to one site where endogenous not exchangeable ADP is bound, and three low affinity sites binding ADP or ATP with a dissociation constant of 15-20 10 il, responsible for the catalytic activity. 

Let us now consider a feasible sequence of events during the cyclic functioning of membrane-bound ATPsynthase in the presence of a transmembrane pH difference. Position 1 in Fig. 5.27 corresponds to the enzyme quasiequilibrium state there are no phosphorylation substrates in the active center, the functional acid groups are protonated due to their contact with the acidic interior of a vesicle (pHj < pK ), and a is open a fast leakage of protons into the external aqueous phase via ATPsynthase is prevented by the barrier hindering the contact of the AH group with the exterior, symbolized in Fig. 5.27 by key b in the locked position. The attachment of phosphorylation substrates to the active center of the coupling factor (transition 1 2)... 

The cyanobacterium Anabaena variabilis ATCC 29413 contains an ATPase which can be distinguished from its coupling factor of phosphorylation. The enzyme appears to be membrane-bound (complete sedimentation by 1 h centrifugation at 100 000 X g), depends on Mg for2activity and is stimulated 3 - 4-fold by micromolar concentrations of Ca (Lockau, Pfeffer 1982). Attempts are reported to determine the intracellular localization and the function of this unusual bacterial enzyme. 

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