Multi-substrate reaction

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A multi-substrate reaction is an enzymatic reaction with several substrates . The treatment of multi-substrate reactions is a branch of enzyme kinetics .

properties

Multi-substrate reaction of LDH with variation of both substrates, pyruvate (A) and NADH + H +

Enzymes normally work with several substrates that are converted into one or more products. The implementation of only one substrate is the exception, e.g. B. Hydrolase and isomerase reactions . Nevertheless, the central equation of enzyme kinetics, the Michaelis-Menten relationship , - strictly speaking - relates to this exceptional case.

As a prototype of an enzymatic conversion, the reaction of lactate dehydrogenase (LDH) will be discussed here, i.e. the conversion of pyruvate with NADH, H + to lactate and NAD + (header in Fig. 1), whereby pyruvate (B) and NADH, H + (A) the role of the two (co-) substrates and lactate (Q) or NAD + (P) play the role of the two products.

The enzyme kinetic parameters (called Michaelis constants or Km values) are determined as follows:

  • the Km value for pyruvate is obtained by varying the pyruvate concentration in the presence of a large excess (saturation concentration) of the second substrate NADH, H + ;
  • the Km value for NADH, H + is determined accordingly by determining the NADH, H + concentration in the presence of a saturating excess of pyruvate.

An alternative is to vary both substrates, that is, to gradually increase the pyruvate concentration in the presence of a different, non-saturating concentration of NADH, H + . Such a procedure has the particular advantage that it also provides information about the reaction mechanism. In part A of the turnover of NADH + H + by the LDH at three non-saturating concentrations of NADH, H + (A3> A2> A1), the Lineweaver-Burk diagram apparently shows the pattern of non-competitive inhibition. In the secondary plot in part B, the axis segments (diamonds) from part A are used to determine the Km value for the second substrate (NADH, H + ).

Sequential and ping-pong mechanisms

Reaction pathways for two-substrate reactions: sequential bi-bi mechanism (top left), ping-pong bi-bi mechanism (bottom left)

Lactate dehydrogenase works according to a "sequential" mechanism. This means that all substrates have to bind to the enzyme before the reaction takes place and the products are released. Since two substrates (A and B) become two products (P and Q), one speaks (in the nomenclature of William Wallace Cleland ) of a “sequential bi-bi” mechanism (Fig. 2A). In the case of an orderly process (´obligatory order´), the binding of the first substrate (A) is necessary so that the enzyme can form the binding point for the second substrate (B), that is, the "lead substrate" (NADH, H + ) must be associated before the subsequent substrate (pyruvate) can bind. Accordingly, the lead product P (NAD + ) must have left the EPQ complex before Q (lactate) can dissociate from EQ. There are basically no complexes of the type EAQ and EBP (substrate and partner product), neither are complexes such as EP or EB. In contrast to this, all substrates / products associate / dissociate in random (´random order´) mechanism in any order. Complexes of the type EB and EP can exist (not shown).

Other enzymes, such as the amino acid transaminases , however, use a “ping-pong-bi-bi” mechanism in which two substrates (bi) are also converted into two products (bi), albeit in separate reaction steps. Transaminases convert their substrate A (amino acid 1) into a product P (alpha-keto acid 1) (ping). Another alpha-keto acid (B) is then taken up and the analogous amino acid transferred (Pong). This mechanism works because the prosthetic group of the enzyme (pyridoxal phosphate) becomes pyridoxamine phosphate between reactions. One speaks of the transition from the enzyme form “E” to the enzyme form “F”. The initial state is restored at the end of the cycle. In the random ping-pong mechanism, the product is released after binding the first substrate molecule (ping). E changes into another enzyme form (F), with which the second substrate reacts to form the second product (Pong).

An analysis as shown in Fig. 1 can be used to differentiate between the two types of reaction:

  • the rate of turnover of pyruvate (B) is investigated in the presence of various (unsaturated) concentrations of NADH, H + (A). Only those enzyme molecules that have already bound NADH, H + can also bind pyruvate (B) and carry out the reaction. The maximum response speed, Vmax, cannot be achieved. However, those enzyme molecules which are present as complex EAB give the expected Michaelis constants (Km) for A and B;
  • in the case of an enzyme according to the ping-pong mechanism, a family of parallel straight lines would be obtained in FIG. 1A (not shown).

Since sequential enzymes are widespread, whereas ping-pong enzymes are less common, only the properties of the first group will be discussed here.

Sequential Mechanisms: Ordered or Arbitrary?

The three basic rules:

  • Using the LDH, the obligatory association of the “lead substrate” A (NADH, H +) in front of the “subsequent substrate” B (pyruvate) was described. As a result, the enzyme can be present as EA and EAB complex, but not as complex EB ( rule 1 ).
  • Similarly, the "lead product" P (NAD + ) has to leave the enzyme before the "secondary product" Q (lactate), that is, an EP complex cannot exist, but an EPQ or EQ complex can ( rule 2 ).
  • Furthermore, there are no “mixed forms” EAQ and EBP, in which a substrate and a product are combined ( rule 3 ).

If enzyme kinetic analyzes are carried out analogously to Fig. 1, but in the presence of a large excess of one of the products (P or Q), the orderly sequence can also be proven, namely by the fact that the maximum reaction rate in the presence of the product (NAD + ) is not achieved (Fig. 3, situation II). With the arbitrary mechanism, however, it would be achieved (Fig. 3 Situation VI). The following scheme is devoted to the possible variations of such an experiment in detail:

Orderly or arbitrary process

Figure 3 shows various kinetics in the presence of a product surplus to distinguish between ordered (´obligatory order´) and arbitrary (´random order´) reaction mechanisms. Capital letters indicate products in excess, a wedge is a substrate, the concentration of which is varied

Variant I: A and B meet E; P can only combine with EQ (rule 2), which implies conversion of B. Increasing [A] accelerates EPQ formation; Vmax is not reached because EPQ dissociation is hindered by P (pattern: " non-competitive ")

Variant II: A and B meet E. P can only combine with EQ, which requires conversion of B., Increasing [B] accelerates EPQ formation; Vmax is not reached because EPQ dissociation is hindered by P (pattern: " non-competitive ")

Variant III: A and B meet EQ. Since there is no EAQ complex (rule 3), A is in competition with Q: increasing [A] lead to the displacement of the interfering neighbor whereby Vmax is reached, but Km increases due to the competition (pattern: " competitive ")

Variant IV: A and B meet EQ. Increasing [B] cannot displace Q, since B could only bind to EA (rule 1). Vmax cannot be achieved because the binding site for A can only be partially saturated (pattern: " non-competitive ")

Variant V: A and B meet EP. Increasing [A] is able to displace the product located in the same binding place, so that Vmax is reached (pattern: " competitive ")

Variant VI: A and B meet EP. Increasing B displaces P from the neighboring binding place, since EPB cannot exist (rule 3). Vmax is achieved because dissociation of Q from EPQ is not hindered by P (pattern: " competitive ")

literature

  • WW Cleland: The kinetics of enzyme-catalyzed reactions with two or more substrates or products . In: Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects . tape 67 , 1963, pp. 104-137 , doi : 10.1016 / 0926-6569 (63) 90211-6 .
  • H. Bisswanger: Enzyme kinetics: theory and methods . 2. edit again Edition. VCH, Weinheim [u. a.] 1994, ISBN 3-527-30032-5 , Chapter 2.6: Multi-substrate reactions .
  • Donald Voet, Judith G Voet, Charlotte W Pratt: Textbook of Biochemistry [with CD-ROM] . Ed .: Annette G. Beck-Sickinger, Ulrich Hahn. 2., act. and exp. Edition. Wiley-VCH, Weinheim 2002, ISBN 3-527-30519-X , Chapter 12: Enzyme kinetics .