Enzymes with two substrates

Most enzyme-catalysed reactions involve two substrates; it is only enzymes catalysing lysis of a molecule or an isomerization reaction (section 2.5) that have only a single substrate.

.2 . (0 -

N

' max

'—

Figure 2.9 The Lineweaver—Burk double-reciprocal plot to determine K and V .

For a reaction involving two substrates (and two products): A + B ^ C + D the enzyme may act by either:

• an ordered mechanism, in which each substrate binds in turn: A + Enz ^ A-Enz

A-Enz + B ^ A-Enz-B ^ C-Enz-D ^ C-Enz + D C-Enz ^ Enz + C

• a ping-pong mechanism in which one substrate undergoes reaction, modifying the enzyme and releasing product, then the second substrate binds, reacts with the modified enzyme and restores it to the original state:

These two different mechanisms can be distinguished by plotting 1/v vs. 1[substrate A] at several different concentrations of substrate B; as shown in Figure 2.10, the lines converge if the mechanism is ordered but are parallel for a ping-pong reaction.

Ordered Ping Pong Lineweaver Burk Graph

Figure 2.10 The Lineweaver—Burk double-reciprocal plots for ordered and ping-pong two-substrate reactions.

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