= Coupled-enzyme readout of glycolytic ATP formation
{c}
The final <substrate-level phosphorylation> in <glycolysis> provides a useful experimental handle: <pyruvate kinase> transfers a <phosphate> group from <phosphoenolpyruvate> to <ADP>, producing <pyruvate> and <ATP>. Adding <lactate dehydrogenase> couples each <pyruvate> formed to consumption of one <NADH>:
$$
\begin{aligned}
\mathrm{phosphoenolpyruvate}+\mathrm{ADP}+\mathrm H^+
&\longrightarrow\mathrm{pyruvate}+\mathrm{ATP},\\
\mathrm{pyruvate}+\mathrm{NADH}+\mathrm H^+
&\longrightarrow\mathrm{lactate}+\mathrm{NAD}^{+}.
\end{aligned}
$$
These equations select free ionic forms consistently: <phosphoenolpyruvate> has charge $-3$, <ADP> $-3$, <ATP> $-4$, and <pyruvate> and <lactate> each $-1$; <NAD+> has one more positive charge than <NADH>. Each line conserves charge. Cancelling <pyruvate> gives one <NADH> consumed per <ATP> formed in this coupled system. A whole-pathway <NADH> measurement has a different meaning, because the earlier <glycolysis> <redox> step produces <NADH>.
The published An Optimized Enzyme-Coupled Spectrophotometric Method for Measuring Pyruvate Kinase Kinetics reports recombinant <pyruvate kinase> prepared in <Escherichia coli>, purified by <chromatography> and <size-exclusion chromatography>. UV-transparent microplate wells contained <ADP>, <phosphoenolpyruvate>, <NADH>, <lactate dehydrogenase> and a <buffer solution> with <magnesium> <ions>. An Epoch plate reader used <spectrophotometry> to follow <absorbance> at $340\,\mathrm{nm}$ every five to ten seconds for one minute at $25\,{}^\circ\mathrm C$. Its Figure 2 presents $A_0-A_t$, increasing as <NADH> is consumed. Heat-inactivated <pyruvate kinase> gave minimal background; a validated active preparation was the positive control. These comparisons support an <enzyme>-dependent readout in the prepared system, rather than a whole-<biological cell> <glycolysis> measurement.<An Optimized Enzyme-Coupled Spectrophotometric Method for Measuring Pyruvate Kinase Kinetics by Saurabh Upadhyay>[Procedure A.5–6 and B.2; Equipment item 1; Figure 2 and caption; Validation of protocol: preparation, optical readout and controls]{ref}
The same report uses $0.5\,\mathrm{cm}$ for optical path length in Procedure B.2.i, but approximately $0.3\,\mathrm{cm}$ in Data analysis §3. An absolute conversion requires the actual path length and sufficient coupling <enzyme> activity; no measured <ATP> rate is inferred here.<An Optimized Enzyme-Coupled Spectrophotometric Method for Measuring Pyruvate Kinase Kinetics by Saurabh Upadhyay>[Procedure B.2.i; Data analysis §3; General notes, items 4 and 6: path-length inconsistency and linear-range conditions]{ref}
For an original dimensional example, stipulate a blank-corrected <absorbance> decline $-dA_{340}/dt=0.024\,\mathrm{min^{-1}}$, a calibrated path length $\ell=0.40\,\mathrm{cm}$ and a chosen calibration coefficient $\varepsilon=6000\,\mathrm{L\,mol^{-1}\,cm^{-1}}$. These are invented teaching inputs, not the protocol's measurements or its quoted coefficient. The <Beer-Lambert law> gives
$$
A_{340}=\varepsilon\ell c_{\mathrm{NADH}},\qquad
-\frac{dc_{\mathrm{NADH}}}{dt}
=\frac{0.024}{6000\times0.40}
=10^{-5}\,\mathrm{mol\,L^{-1}\,min^{-1}}.
$$
In a stipulated $0.20\,\mathrm{mL}$ reaction <volume (thermodynamics)>, this is $2\,\mathrm{nmol\,min^{-1}}$ of <NADH> consumed and, conditionally, $2\,\mathrm{nmol\,min^{-1}}$ of <ATP> formed. The conditional inference requires rapid <pyruvate> consumption by the coupling <enzyme>, no other <NADH>-consuming <chemical reactions>, and a linear, correctly calibrated optical response. The readout measures this last <glycolysis> step; it cannot establish the full two-net-<ATP>-per-<glucose> balance by itself.
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