Slow‐binding inhibition: A theoretical and practical course for students

Abstract

Tyrosinase (EC 1.14.18.1) catalyzes the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) to 2,3,5,6-tetrahydro-5,6-dioxo-1H-indole-2-carboxylate (dopachrome), according to the classical Michaelis-Menten kinetic mechanism. The enzyme is strongly but slowly inhibited by ␣-amino-␤-[N-(3-hydroxy-4-pyridone)] propionic acid (L-mimosine), a toxic plant amino acid. Easily available reagents and simple spectrophotometric detection of the product make the experimental characterization and kinetic analysis of tyrosinase action convenient and interesting for teaching purposes. In the present article, we present a theoretical and practical guide to the kinetic analysis of slow-binding inhibition. The effect of L-mimosine on tyrosinase is established by progress curve measurements, carried out on a conventional spectrophotometer equipped with a rapid kinetic accessory. In the analysis, we recommend a classical linearization approach but we also took advantage of a more reliable nonlinear regression method to avoid subjective bias. A multistep procedure starts by careful inspection of the curves to discriminate between candidate mechanisms. Next, the evaluation of initial and steady-state velocities provides information on the enzyme catalytic and Michaelis-Menten constants, as well as the corresponding inhibition constants. Subsequently, an appropriate mathematical derivation enables estimation of the isomerization rate constants characteristic for a slow-binding inhibitor. To conclude, we suggest simultaneous multivariable regression, using all the progress curve data, to cross-check the proposed reaction mechanism and evaluated kinetic constants.

Antifolate Activity of Epigallocatechin Gallate against Stenotrophomonas maltophilia

“…The calculated K m and k cat values at pH 7.4 were essentially the same for the enzymes from the two isolates and similar to those of DHFRs from other species (3,40). S. maltophilia DHFR was strongly inhibited by MTX (inhibition constant [K i ], 18 pM for isolate 1), showing slow-binding inhibition (12) as described for the inhibition of DHFRs from other biological sources by this compound (38). However, because MTX is not used clinically in the treatment of S. maltophilia-related diseases, this inhibition has not been further characterized.…”

Synthesis and Preliminary In Vitro and In Vivo Evaluation of Thiirane‐Based Slow‐Binding MMP Inhibitors as Potential Radiotracers for PET Imaging

“…The enzyme kinetics of the aforementioned mechanism can be described by a rapid second‐order rate constant for the onset of slow‐binding inhibition ( k on ) resulting in a gelatinase inhibitor complex that cannot readily reverse itself as manifested in a slow first‐order rate constant ( k off ). The ratio k on / k off represents the dissociation constant K i of the slow‐binding inhibition complex, whereas the reciprocal of the dissociation rate constant (1/ k off ) expresses the inhibitor‐enzyme complex residence time τ, the duration in which the inhibitor is physically bound to the target . Compared to the short residence times (in the order of seconds) of other competitive MMPIs bound to the gelatinases, the complexes of MMP‐2 or MMP‐9 with SB‐3CT and its derivatives exhibit long residence times (in the order of minutes).…”

Use of mushroom tyrosinase to introduce michaelis‐menten enzyme kinetics to biochemistry students

“…This gives them an experience similar to that which they would encounter in a research lab were they working with an enzyme that had not been characterized. Also, most published procedures do not provide an easy way for students to fit the Michaelis‐Menten equation to their data in order to obtain values for V max and K m . Here a Microsoft Excel template is provided to students which enables them to fit the Michaelis‐Menten equation to their data in order to accurately determine values for V max and K m .…”

“…For several reasons, most published lab exercises using mushroom tyrosinase were not suitable for our students or our laboratory course. Some procedures are presented at a relatively advanced level (perhaps for second‐semester biochemistry students or honors students who already have some lab experience with enzyme kinetics) and require lab periods longer than 3 hours . Some procedures require equipment which may not be available in a typical undergraduate teaching laboratory (e.g., a microtiter plate reader or stopped‐flow spectrophotometer) .…”

“…Some procedures are presented at a relatively advanced level (perhaps for second‐semester biochemistry students or honors students who already have some lab experience with enzyme kinetics) and require lab periods longer than 3 hours . Some procedures require equipment which may not be available in a typical undergraduate teaching laboratory (e.g., a microtiter plate reader or stopped‐flow spectrophotometer) . Our procedure is presented at a level designed for first‐semester biochemistry lab students who are studying enzyme kinetics for the first time in the accompanying lecture course.…”