2.2.1 Recycling of Cofactors
The major and crucial distinction between redox enzymes and hydrolases described
in the previous chapter, is that the former require redox cofactors, which donate or
accept the chemical equivalents for reduction (or oxidation). For the majority of
redox enzymes, nicotinamide adenine dinucleotide [NAD(H)] and its respective
phosphate [NADP(H)] are required by about 80% and 10% of redox enzymes,
respectively. Flavines (FMN, FAD) and pyrroloquinoline quinone (PQQ) are
encountered more rarely. The nicotinamide cofactors – resembling ‘Nature’s complex hydrides’ – have two features in common, i.e., they are relatively unstable
molecules and they are prohibitively expensive if used in stoichiometric amounts.
24
In addition, they cannot be replaced by more economical man-made substitutes.
Since it is only the oxidation state of the cofactor which changes during the
reaction, while the remainder of the complex structure stays intact, the cofactor
may be regenerated in situ by using a second concurrent redox-reaction to allow it
to re-enter the reaction cycle. Thus, the expensive cofactor is needed only in
catalytic amounts, which leads to a drastic reduction in cost. The efficiency of
such a recycling process is measured by the number of cycles which can be
achieved before a cofactor molecule is finally destroyed. It is expressed as the
‘total turnover number’ (TTN, Sect. 1.4.3) – which is the total number of moles of
product formed per mole of cofactor during its entire lifetime.
25 As a rule of thumb,
a few thousand cycles (10
3 –10
4 ) are sufficient for redox reactions on a laboratory
scale, whereas for technical purposes, total turnover numbers of at least 10
5 are
highly desirable. The economic barrier to large-scale reactions posed by cofactor
O
O
N
NH
N
N
H
H
O
O
N
NH
N
N
H H O
NH 2
N
O
NH 2
N
O
R 2
R 1
R 3
R
2
R
1
EWG
R
2
R
1
EWG
H
H
R
3
H
H
EWG
R 1
R
2
R
3
OH
R 2
R 1
OH
R 2
R 1
Cofactors: Nature's Complex Hydrides
- [H
- ]
Flavin
Nicotinamide
- [H
- ]
Ene-Reductase
or
chiral products
prochiral
substrate
or
or
Recycling System
Dehydrogenase
Cofactor
Cofactor-H 2
*
*
*
*
*
*
Scheme 2.107 Reduction reactions catalyzed by dehydrogenases (EWG electron withdrawing
group)
24 The current prices for 1 mole are: NAD
+ US $1400, NADH US $2600, NADP
+ US $18,000,
NADPH US $70,000.
25 In cofactor recycling the TTN is sometimes called ‘cycle number’.
134
2 Biocatalytic Applications
The major and crucial distinction between redox enzymes and hydrolases described
in the previous chapter, is that the former require redox cofactors, which donate or
accept the chemical equivalents for reduction (or oxidation). For the majority of
redox enzymes, nicotinamide adenine dinucleotide [NAD(H)] and its respective
phosphate [NADP(H)] are required by about 80% and 10% of redox enzymes,
respectively. Flavines (FMN, FAD) and pyrroloquinoline quinone (PQQ) are
encountered more rarely. The nicotinamide cofactors – resembling ‘Nature’s complex hydrides’ – have two features in common, i.e., they are relatively unstable
molecules and they are prohibitively expensive if used in stoichiometric amounts.
24
In addition, they cannot be replaced by more economical man-made substitutes.
Since it is only the oxidation state of the cofactor which changes during the
reaction, while the remainder of the complex structure stays intact, the cofactor
may be regenerated in situ by using a second concurrent redox-reaction to allow it
to re-enter the reaction cycle. Thus, the expensive cofactor is needed only in
catalytic amounts, which leads to a drastic reduction in cost. The efficiency of
such a recycling process is measured by the number of cycles which can be
achieved before a cofactor molecule is finally destroyed. It is expressed as the
‘total turnover number’ (TTN, Sect. 1.4.3) – which is the total number of moles of
product formed per mole of cofactor during its entire lifetime.
25 As a rule of thumb,
a few thousand cycles (10
3 –10
4 ) are sufficient for redox reactions on a laboratory
scale, whereas for technical purposes, total turnover numbers of at least 10
5 are
highly desirable. The economic barrier to large-scale reactions posed by cofactor
O
O
N
NH
N
N
H
H
O
O
N
NH
N
N
H H O
NH 2
N
O
NH 2
N
O
R 2
R 1
R 3
R
2
R
1
EWG
R
2
R
1
EWG
H
H
R
3
H
H
EWG
R 1
R
2
R
3
OH
R 2
R 1
OH
R 2
R 1
Cofactors: Nature's Complex Hydrides
- [H
- ]
Flavin
Nicotinamide
- [H
- ]
Ene-Reductase
or
chiral products
prochiral
substrate
or
or
Recycling System
Dehydrogenase
Cofactor
Cofactor-H 2
*
*
*
*
*
*
Scheme 2.107 Reduction reactions catalyzed by dehydrogenases (EWG electron withdrawing
group)
24 The current prices for 1 mole are: NAD
+ US $1400, NADH US $2600, NADP
+ US $18,000,
NADPH US $70,000.
25 In cofactor recycling the TTN is sometimes called ‘cycle number’.
134
2 Biocatalytic Applications