The calculations of net gain of ATP can be made only on certain assumptions
(i) There is a sequential, orderly pathway functioning, with one substrate forming the next and with glycolysis, TCA cycle and ETS pathway following one after another.
(ii) The NADH synthesised in glycolysis is transferred into the mitochondria and undergoes oxidative phosphorylation.
(iii) None of the intermediates in the pathway are utilised to synthesise any other compound.
(iv) Only glucose is being respired-no other alternative substrates are entering in the pathway at any of the intermediary stages.
But this kind of assumptions are not really valid in a living system. All pathways work simultaneously and do not take place one after another. Substrates enter the pathways and are withdrawn from it as and when needed; enzymatic rates are controlled by multiple means. In over all steps, there is a net gain of 36 ATP molecules during aerobic respiration of one molecule of glucose.