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A competitive inhibitor is an enzyme inhibitor that produces competitive inhibition relative to a specified substrate. In the usual single-substrate kinetic description, it reduces the low-substrate response while leaving the limiting rate unchanged. A common molecular model has the inhibitor bind free enzyme and prevent formation of the enzyme-substrate complex; the kinetic classification alone does not prove a particular binding location.^[ref]

For the simple reversible model at fixed free inhibitor concentration $[I]$,Here $K_i$ is the inhibition constant for this model and $K_M$ the uninhibited Michaelis constant. Take illustrative values $V_{\max }=12\mu \mathrm{M}\mathrm{mi}{\mathrm{n}}^{-1}$, $K_M=2\mu \mathrm{M}$, $K_i=5\mu \mathrm{M}$ and $[I]=10\mu \mathrm{M}$. Then $\alpha =3$ and the apparent Michaelis constant is6micromolar. At $[S]=2\mu \mathrm{M}$, the inhibited rate is3micromolar per minute, compared with6without inhibitor.

As substrate concentration grows, both rates approach the same limiting value in this model. That is a limit, not an assertion that a particular finite substrate addition removes all inhibition. Characterizing a candidate therefore requires rates over suitable substrate and inhibitor ranges with controlled enzyme amount; one reduced rate does not establish that the inhibitor is competitive.^[ref]

An enzyme inhibitor lowers an enzyme's reaction rate. In reversible enzyme inhibition, reducing free inhibitor concentration can restore activity; slow release can make this difficult to distinguish experimentally from irreversible inactivation. Competitive inhibition and mixed inhibition describe different changes in enzyme kinetics; pure noncompetitive inhibition is a special case of mixed inhibition, not a name for every inhibitor that binds away from an active site.^[ref]

The useful question is how adding substrate changes the inhibition. The model below connects binding to a measurable reaction rate, predicts when more substrate can overcome inhibition, and supplies examples for comparison with an enzyme inhibition initial-rate assay. Its inhibition constants are explicitly defined; neither a binding cartoon nor one reduced reaction rate by itself determines a mechanism.

Malonate is the dianion of malonic acid, with two carboxylate groups separated by one methylene group. Its three-carbon skeleton resembles part of the four-carbon skeleton of succinate.

Malonate is a classical inhibitor of succinate dehydrogenase: structural similarity allows binding without the normal substrate transformation. The example illustrates how an enzyme inhibitor can probe the contribution of a particular reaction to a metabolic pathway.

A trypsin colorimetric assay follows enzyme activity through absorbance at $405\mathrm{nm}$. Trypsin cleaves an amide bond in the synthetic arginine derivative BAPNA, releasing p-Nitroaniline.^[ref]

The inspected Sigma procedure prepares fresh trypsin in cold $1\mathrm{mM}$ hydrochloric acid and fully dissolves $1mg/ml$ BAPNA. Its buffer solution stock contains $200\mathrm{mM}$ triethanolamine and $20\mathrm{mM}$ calcium chloride, at pH $7.8$. Three $3.00\mathrm{ml}$ cuvettes compare trypsin, trypsin plus the enzyme inhibitor aprotinin, and a blank replacing trypsin with its acid solvent. Sodium chloride solution replaces aprotinin in the uninhibited control. A thermostatted instrument for spectrophotometry holds $25{}^{\circ }\mathrm{C}$ and a $1\mathrm{cm}$ light path. After equilibration, adding BAPNA starts approximately five minutes of absorbance recording. The uninhibited sample checks activity; the blank checks background; the inhibited sample tests suppression.^[ref]

Let $s_U$, $s_I$ and $s_B$ denote the measured absorbance slopes per minute over the same linear time interval. Blank subtraction gives $s_U-s_B$ and $s_I-s_B$. If p-Nitroaniline accounts for the changing absorbance, the Beer-Lambert law givesHere $c$ is product molar concentration and $l$ is the geometric light path through the chemical solution. Sigma uses $\epsilon =9.96\mathrm{m}{\mathrm{M}}^{-1}\mathrm{c}{\mathrm{m}}^{-1}$.^[ref]

Choose original illustrative slopes $s_U=0.1036$, $s_I=0.0538$ and $s_B=0.0040\mathrm{mi}{\mathrm{n}}^{-1}$. The corrected uninhibited slope is $0.0996\mathrm{mi}{\mathrm{n}}^{-1}$, giving $dc/dt=0.0100\mathrm{mM}\mathrm{mi}{\mathrm{n}}^{-1}=10.0\mu \mathrm{M}\mathrm{mi}{\mathrm{n}}^{-1}$ at $l=1\mathrm{cm}$. In $3.00\mathrm{ml}$, the product amount of substance increases at $0.0300\mu \mathrm{mol}\mathrm{mi}{\mathrm{n}}^{-1}$. The corrected inhibited slope is $0.0498\mathrm{mi}{\mathrm{n}}^{-1}$, half as large. The relative suppression is thereforeThe background cancels in the numerator, but remains essential in the denominator. Using uncorrected slopes would instead give about $48.07\%$. Differentiating $A_{405}=\epsilon lc$ with fixed $\epsilon$ and $l$ explains the rate conversion; dimensional analysis checks it independently, since $\mathrm{mi}{\mathrm{n}}^{-1}/(\mathrm{m}{\mathrm{M}}^{-1}\mathrm{c}{\mathrm{m}}^{-1}\mathrm{cm})=\mathrm{mM}\mathrm{mi}{\mathrm{n}}^{-1}$.

The inference is a reaction rate on BAPNA under the stated conditions, rather than a direct count of trypsin molecules or proof of every cut in a protein. Cloudiness, other absorbing solutes, or different product protonation can compromise the Beer-Lambert law interpretation. A matched blank and a linear interval are therefore part of the measurement's meaning. A coloured enzyme inhibitor may require its own matched blank; a changed substrate or pH requires a fresh calibration. The constructed constant slopes above describe only that interval, not indefinite product accumulation.