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MiniReview
a;!
Faculty of Technology, The University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Yugoslavia
b
Faculty of Science, The University of Novi Sad, 21000 Novi Sad, Yugoslavia
Received 7 February 2002; received in revised form 1 August 2002 ; accepted 2 August 2002
First published online 18 September 2002
Abstract
This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing
alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in bakers yeast, Saccharomyces cerevisiae. The opening section deals with the
substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the
kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary
structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known
artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of
action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the
bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate
specificity and the enantioselectivity of the yeast enzyme.
! 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Keywords : Yeast alcohol dehydrogenase; Alcohol dehydrogenase ; Saccharomyces cerevisiae
1. Introduction
Yeast alcohol dehydrogenase (EC 1.1.1.1) is a member
of a large family of zinc-containing alcohol dehydrogenases. The primary structures of 47 members of this family
have been determined and aligned, and an evolutionary
tree has been constructed, assuming a divergent evolution
from a common ancestral gene [1]. In this way, it was
possible to identify four divergent groups of alcohol dehydrogenases in this family: vertebrates, plants, eukaryotic
microorganisms and prokaryotic bacteria. Bakers yeast
(Saccharomyces cerevisiae), a member of the third group,
has three isoenzymes of alcohol dehydrogenase : YADH-1,
YADH-2, and YADH-3. YADH-1 is the constitutive form
2. Isoenzymes of YADH
Yeast alcohol dehydrogenase was one of the rst enzymes to be puried and isolated [8]. If the steady-state
kinetic properties of the ADH isoenzymes are compared, a
large degree of similarity is detected. Table 1 shows the
steady-state kinetic constants for the three isoenzymes of
YADH, isolated from bakers yeast.
1567-1356 / 02 / $22.00 ! 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
PII: S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 7 - 5
482
Table 1
Steady-state kinetic constants of yeast ADH isoenzymes with ethanol
and acetaldehyde as substrates, at pH 7.3, 30Ca
Constant
Unit
YADH-1
YADH-2
YADH-3
V1
KA
KB
V1 /KB
V2
KQ
KP
V2 /KP
s31
WM
mM
mM31 s31
s31
WM
mM
mM31 s31
340
170
17
20
1700
110
1.1
1540
130
110
0.81
160
1040
50
0.09
11 550
450
240
12
37.5
2100
70
0.44
4770
3. Substrate specicity
Yeast alcohol dehydrogenase catalyzes the following reversible redox reaction [5]:
Table 2
Steady-state kinetic constants for the oxidation of various alcohols at neutral pH
Constant
Unit
Ethanola
Propan1-ola
Butan1-ola
Hexan1-olb
Decan1-olb
Propan2-olc
(S)-(+)-Butan2-olc
Allyl
alcohold
Ethyleneglycold
Trise
V1
KA
KiA
KB
V1 /KA
V1 /KB
0.0001
V1 KiA /KA
Keq f
s31
WM
WM
mM
mM31 s31
mM31 s31
454
109
325
21.7
4165
20.9
67
150
235
29.2
447
22.9
25
250
160
32
100
0.78
15.4
169
152
3.2
91
4.8
14.4
200
190
0.1
72
144
7
597
378
117
11.7
0.06
0.9
376
398
35
2.4
0.026
546
520
730
14.6
1058
37.5
7.0
370
550
444
19.2
0.016
0.5
698
842
6415
0.72
s31
^
1354
0.00019
105
^
16
0.00027
13.8
^
13.7
^
4.4
0.146
0.95
0.40
766
^
10.4
^
0.60
^
483
Unit
Acetaldehydea
Butyraldehydea
Acetoneb
Butan-2-one-b
Chloroacetaldehydec
NDMAd
DACAe
V2
KQ
KiQ
KP
V2 /KQ
V2 /KP
V2 KiQ /KQ
s31
WM
WM
mM
mM31 s31
mM31 s31
s31
3850
96
12.5
0.9
40100
4280
501
3450
97
7
27.5
35570
125
249
9
43
17.5
477
209
0.019
3.66
0.7
38
15.2
285
18.4
0.0025
0.38
117
270
74
4
431
25.2
31.9
2.1
456
119
1.5
4.5
1.4
0.54
0.176
46
7.6
0.61
3.8
0.29
0.03
Calculated
Calculated
c
Calculated
d
Calculated
e
Calculated
b
from
from
from
from
from
the data of
the data of
the data of
the data of
the data of
V0
k 9 k 5 k7
!
"
1 Xk1 k14
k12 k13 A k11 B
1
W
k1 k9 k13
k12 k13 A k13 =k1 k11 B A
!
"
1 Xk4
k12 k13 A
1
k3 k 9
k12 k13 A k13 =k1 k11 B B
!
"
1 Xk4
k12 k13 A k11 B
1
W
k3 k 9
k12 k13 A k13 =k1 k11 B AB
Scheme 1.
484
Table 4
Thermodynamics of the yeast alcohol dehydrogenase reaction, at pH 7.0, 25C [45]
k1
k31
k32
k9
k5
k7
k2
k41
k42
k10
k6
k8
E3EA3EA3EAB3EPQ3EQ3E
Rate constant
vG0 (kJ/mol)
Dissociation constant
31
31
k1
k2
k4 /k3
WM
s31
WM
k9
k10
k5
k6
k7
k8
s31
s31
s31
WM31 s31
s31
WM31 s31
7 " 0.2
2100 " 57
158 000
(11)
(3900)a
(^)a
3980 " 97
35 040 " 870
10 900 " 160
5.0 " 0.04
388 " 5
28.1 " 0.5
(4000)a
(35 000)a
(11 000)a
(4.3)a
(480)a
(44)a
k2 /k1
WM
300
320.08
k41 /k31
k42 /k32
k10 /k9
^
WM
75b
2110c
8.75
10.70
315.26
5.37
k5 /k6
WM
2180
15.18
k7 /k8
WM
13.80
27.73
Total
Keq = 0.000068d
23.64
23.7
Data in parentheses are from the steady-state kinetic measurements of Dickinson and Dickenson [31], at pH 7.0, 25C.
Taken from Northrop [46].
c
Calculated from the equilibrium constant: k4 /k3 = (k41 /k31 )/(k42 /k32 ).
d
Calculated from the Haldane relationship : Keq = V1 KiQ Kp /(V2 KiA KB ).
b
k2 k4 k10
k8 Q
k6 k8 k10 PQ
V0
k6 k10 P
6
where Y = 1+k9 /k4 .
Eq. 6 satises the results obtained for the reduction of
acetaldehyde and butyraldehyde in predicting a linear reciprocal equation, in which the KiQ , V2 /KQ and V2 KiQ /KQ
constants are independent of the nature of the aldehyde
(Table 3).
The kinetic mechanism in Scheme 1 is compatible with
deuterium isotope eects on maximal rates reported for
ethanol, D V1 = 1.8, D V1 /KA = 1.8, and D V1 /KB = 3.2 [39],
propan-1-ol, D V1 = 3.7 [40], butan-1-ol, D V1 = 3.7 [41],
and propan-2-ol, D V1 = 2.2 around neutrality [13]. With
ethanol, the eect on D V1 /KA was smaller than on D V1 /
KB , suggesting that NAD binds before ethanol; the still
signicant size of D V1 /KA is probably due to dissociation
of NAD from the ternary complex [39].
With propan-2-ol and acetone, the kinetic mechanism is
steady-state random in both directions [13]. A similar kinetic mechanism probably holds for most branched and
secondary alcohols [34].
5. Pre-steady-state kinetics
Pre-steady-state kinetic studies provide the numerical
values of the rate constants in the mechanism. The presteady-state kinetics of yeast alcohol dehydrogenase has
6. Primary structure
YADH-1 is a tetramer, composed of four identical subunits ; each subunit consists of a single polypeptide chain
with 347 amino acids, with a molecular mass of 36 kDa
[47]. Each subunit has one coenzyme-binding site and one
rmly bound zinc atom, which is essential for catalysis
[50,51] ; the catalytic domain provides the ligands to this
zinc atom: Cys-46, His-67 and Cys-174. The second zinc
atom/subunit is liganded in a tetrahedral arrangement by
four sulfur atoms from the cysteine residues 97, 100, 103
and 111; this zinc atom only has a structural role [52].
Table 5 shows the primary structures of the three isoenzymes of YADH [4,53^55]. The alignment of amino acid
residues for all 47 members of the ADH family was made
progressively rather than pairwise [1].
Table 5
Primary structure of the three isoenzymes of yeast alcohol dehydrogenase
485
drogenase and YADH-1 are homologous, and the homology amounts to 25% of the amino acid residues [5].
YADH-1 has been crystallized, but only preliminary crystallographic studies have been reported [56]. The threedimensional structure of horse liver alcohol dehydrogenase
in several binary and ternary complexes with coenzymes,
substrates and inhibitors has been solved at high resolution [47]. The tertiary structures of liver and yeast enzyme
are highly similar and able to accommodate extensive sequence changes between the enzymes [57].
Analogous to the liver enzyme, the subunits of the yeast
enzyme are probably divided into two domains : the catalytic domain and the coenzyme-binding domain. The two
domains are unequal in size ; the catalytic domain contains
3/5 of all amino acids, whereas the coenzyme-binding domain contains the remaining 2/5 of the amino acids. The
domains are separated by a cleft, containing a deep pocket
which accommodates the substrate and the nicotinamide
moiety of the coenzyme. One domain binds the coenzyme
and the other provides ligands to the catalytic zinc, as well
as to most of the groups that control substrate specicity
[47].
Since the liver and yeast enzymes are homologous, molecular modeling of the yeast enzyme can approximate the
structure of one subunit, but not yet the quaternary arrangement [57].
Fig. 1 shows a model of the active site of the yeast
enzyme, drawn schematically after a model obtained in a
molecular graphics display system by Plapp et al. [58]. The
3D-model of the active site of YADH provides an illustration of the main working machinery of the yeast enzyme. In order to perform catalysis, the active site of the
enzyme has to bind a molecule of substrate and a molecule
of coenzyme in a productive mode, and, subsequently catalyze a hydride-transfer reaction between them.
The adenosine-binding site is easily accessible from solution, whereas the nicotinamide-binding site is situated at
the center of the molecule, buried deep inside the protein
[47]. Numerous amino acid residues in the primary structure of the enzyme are involved in substrate and coenzyme
binding and in catalysis (Table 6).
7.1. Substrate-binding pocket
The numbering of amino acids corresponds to horse liver alcohol dehydrogenase; alignment and numbering of amino acid in the yeast isoenzymes according to Sun and Plapp [1].
486
Table 6
Positions of residues that participate in enzymatic functions of the yeast
enzyme (adopted from Jornval et al. [57])
Adenine binding pocket
Interior
Ser-198
Ile-250
Ile-222
Ser-269
Gly-224
Ala-274
Phe-243
Ala-277
Surface
Ser-271
Ala-273
Adenosine^ribose binding
Gly-199
Lys-228
Asp-223
Ser-269
Gly-225
Pyrophosphate binding
His-47
Leu-203
Gly-202
Nicotinamide ribose
Gly-293
Ser-269
Nicotinamide
Thr-178
Substrate-binding pocket
Trp-57
Thr-141
Trp-93
Met-294
Asn-110
Ala-296
Leu-132
Ile-318
Tyr-140
Proton relay system
Thr-48
His-51
487
The three yeast ADH genes have been cloned and described [4,54,55]. Therefore, it was possible to change individual amino acids in the primary structure of YADH-1
via site-directed mutagenesis and isolate a quantity of mutated enzymes (Table 7).
In recent years, a number of genetically engineered mutants of YADH-1 were isolated and kinetically characterized, principally by Plapp and his co-workers. Most of
these mutations involve amino acids which are intimately
involved in the binding of substrates and in catalysis, and
provide information about the general principles concerning the function of the catalytic residues. Table 7 shows
the steady-state kinetic properties of all YADH mutants
described so far participating in substrate binding and in
catalysis.
Table 7
Steady-state kinetic constants for YADH mutants, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30C
Mutant
Source
YADH-1
Substrate-binding pocket
Met294Leu
Trp57Met
Trp57Leua
Trp93Ala
Ligands to the active-site zinc
Asp49Asn
Glu68Gln
The proton relay system
Thr48Ser
Thr48Ser :Trp93Ala
Thr48Ser :Trp57Met:Trp93Ala
Thr48Cys
Thr48Ala
His51Gln
His51Glu
340
2000
20
1700
15 500
1545
[9]
500
220
99
110
794
265
91
48
26.3
4.9
7.4
0.07
2100
1900
211
ND
26 250
6 790
2 245
ND
2100
513
ND
ND
[9]
[20]
[19]
[20]
7.5
9.9
0.83
24
0.02
0.24
113
730
125
4 560
2.3
13
[39]
[39]
200
140
120
61
61
27
2
2200
11.8
152
0.033
22.2
0.75
No detectable activity
No detectable activity
245
1.4
26
0.26
1500
530
ND
13 640
4 077
ND
2027
5.7
ND
2800
ND
25 450
ND
215
ND
[20]
[20]
[20]
[58]
[58]
[61]
[58]
ND = not determined.
a
Determined at pH 8.2, 25C.
488
9. Binding of coenzymes
Fig. 2 summarizes the steady-state and ligand-binding
data relevant for the binding of coenzymes to the free
enzyme.
The dissociation constant of the EWNAD complex for
the yeast enzyme, K
E;NAD , is practically pH-independent;
on the other hand, the dissociation constant of the
EWNADH complex, KE;NADH , decreases with lower pHvalue over three apparent pKa values (6.6, 8.0 and 9.0).
The association rate constant for the binding of NADH
489
Table 8
Steady-state kinetic constants for YADH mutants in the adenylate binding pocket, with ethanol and acetaldehyde as substrates, determined at pH 7.3,
30C
Mutant
V1 (s31 )
V2 (s31 )
Source
YADH-1
Adenine site substitutions
Ser198Phe
Gly224Ile
Gly225Arg
Adenosine^ribose binding
Asp223Gly
Asp223Gly:Gly225Arg
Pyrophosphate binding
Leu203Alaa
Leu203Ala :Thr178Sera
His47Arg
Gly204Ala
Ala200v:Ala201Leub
NMN^ribose binding
Ser269Ile
340
2000
20
1700
15 500
1 545
[9]
40
360
550
1.25
414
1222
0.14
16
21
150
4000
2400
25
6 060
12 000
71
20 000
18 000
[62]
[62]
[62]
38
17
2.1
0.94
0.2
0.13
300
110
60
18.33
75
20
[63]
[63]
106
31.9
60
8
67
56.4
61.3
400
0.26
13.13
ND
ND
0.9
0.02
1.4
ND
ND
460
200
ND
ND
ND
46 000
25
ND
ND
ND
98
11
ND
[64]
[64]
[32]
[62]
[62]
1.0
0.36
0.003
5.4
13.85
0.31
[62]
ND = not determined.
a
Determined at pH 8.2, 25C.
b
Alignment of amino acids according to Table 5. Ala200 is an insertion in the yeast enzyme with respect to other members of the alcohol dehydrogenase family of enzymes; therefore, this residue is not counted in the primary structure that follows after this residue [1].
tions in the yeast enzyme, outlined in the preceding sections, strongly suggest that the integrity of the proton
relay system is indispensable for the activity of the enzyme.
Based on this integrity of the relay system, which is
maintained throughout the catalytic cycle, Cook and Cleland [60] have proposed the chemical mechanism of action
for the yeast enzyme as shown in Scheme 2.
In this mechanism, B and P represent alcohol and ketone, and k3 , k4 , k5 and k6 represent hydride-transfer
steps ; X is an intermediate with the stoichiometry of an
alkoxide, and k1 and k2 are the steps in which a proton is
transferred from B to a group on the enzyme to give X,
and similarly for the reverse process.
An assignment of appropriate pKa values to all dissociation forms of the enzyme in Scheme 2 was founded on
studies of the pH dependence of the steady-state kinetics
and ligand-binding parameters [14,16,26,33^36,65], as outlined below.
Table 9 shows the macroscopic pKa values calculated
from the pH proles of the maximal rates (V1 ) and the
specicity constants (V/K) with various substrates.
Table 10 presents the pKa values calculated from the pH
proles of binding constants (Ki ) for competitive dead-end
inhibitors.
Scheme 2.
The specicity constants V/K with nonsticky substrates, such as propan-1-ol, propan-2-ol, NDMA,
DACA and acetone, provide information on catalytically
active groups in enzyme^coenzyme complexes [66], if the
pH proles of V/K are tted to initial-rate equations appropriate to the mechanism in Scheme 2 [36]. In this way,
the pK1 (8.0) and pK5 (7.9^8.0) values in Scheme 2 were
estimated. From the binding of azide, a dead-end inhibitor
competitive with alcohols, the value for pK1 (7.9) was
conrmed; from the binding of acetamide, a dead-end
inhibitor competitive with aldehydes, the values for pK4
(8.3) and pK5 (7.9) were estimated.
pH proles for the V1 function provide information on
catalytically active groups in the productive ternary enzymeWNAD Walcohol-complex [66]. In this way the pK2
value was estimated (8.3), from the pH proles of V1
with butan-1-ol and propan-2-ol.
An indirect estimation provided the value of pK3 (8.3)
[36].
The chemical mechanism of action, presented in Scheme
1, can be drawn entirely in terms of the proton relay
system, as is shown in Fig. 3; in Fig. 3, however, the
Thr-48 residue was omitted from the relay for the sake
of simplicity. The key feature of Fig. 3 is that His-51 lies
at the surface of the protein and thus can be deprotonated
as in the conversion of HEAX to EAX or HEQP to EPQ,
while reactants are bound and the state of protonation of
molecules in the substrate-binding site is locked. Thus,
HEAX can be deprotonated to EAX without preventing
subsequent hydride transfer.
A dierent view on the chemical mechanism of action of
yeast alcohol dehydrogenase has been presented by Branden et al. [5]. These authors proposed that the Zn2 -bound
490
Table 9
Macroscopic pKa values and pH-independent limiting constants in various YADH-catalyzed reactions (adopted from Leskovac et al. [36])
Substrate
pKa
Butan-1-ol
V1 (s31 )
6.1
7.3
8.3
Propan-2-ol
6.2
7.4
V1 /KB (s31 )
8.3
Propan-1-ol
6.7
V1 /KB (mM31 s31 ) 7.4
8.2
Propan-2-ol
6.5
7.1
V1 /KB (M31 s31 )
7.8
Acetone
7.9
V1 /KB (M31 s31 )
8.2
9.0
DACA
8.0
V2 /KP (mM31 s31 )
8.0
NDMAa
V2 /KP (mM31 s31 )
Limiting constant
Dixon^Webb plot
191
increases with pH
81
increases with pH
9.0
increases with pH
155
increases with pH
6.9
decreases with pH
0.25
decreases with pH
2.2
0.9
plateau at low pH
plateau at high pH
Complex
pKa
Limiting constant
7.9
8.3
491
namic and kinetic criteria, the most sensitive one for detection of tunneling is the breakdown of the above relationship, particularly for the secondary kinetic isotope
eect [73]. In line with this, Cha et al. [74] have studied
the oxidation of benzyl alcohol by NAD , using six dierently labeled alcohols as substrates :
14 C(C 6 H 5 CH 2 OH; 14 C(C 6 H 5 CD2 OH;
C 6 H 5 CH; TOH; C 6 H 5 CT; HOH;
C 6 H 5 CD; TOH; C 6 H 5 CT; DOH
in order to obtain all combinations of isotope eects. This
reaction is suitable for exploring hydrogen tunneling because of the lack of any kinetic complexity, as it has a
rate-limiting H-transfer step [75].
Cha et al. [74] have demonstrated that, in this reaction,
the exponents in Eq. 8 are 3.58 and 10.2 for the primary
and secondary kinetic isotope eect, respectively, indicating signicant breakdown of the semi-classical upper limit.
For hydrogen tunneling to occur, the reactive carbon
atoms have been brought close together so that the classical energy barrier is penetrated. Thus, it appears that
hydrogen tunneling is an additional general phenomenon
which facilitates the YADH catalysis [23,76^78].
Leskovac et al. [79] have studied the primary kinetic
isotope eects and the internal thermodynamics of the
YADH-catalyzed oxidation of 2-propanol-h8 and 2-propanol-d8 with NAD ; the properties of this reaction were
compared with non-enzymatic model redox reactions of
N1 -substituted-1,4(1 H2 )dihydronicotinamides and N1 -substituted-1,4(1 H2 H)dihydronicotinamides with a number of
various oxidizing agents. The kinetic and thermodynamic
properties of the enzymatic reaction closely resemble the
model hydride-transfer reactions which probably proceed
via a linear transition state, and are very dissimilar from
492
[8]
[9]
[10]
[11]
[12]
[13]
Fig. 5. Stereospecicity of YADH catalysis. NADH binds anti, presenting Re-hydrogen (HRe ) to acetaldehyde lying above the coenzyme in this
diagram. For clarity, Thr-178 is not shown; the methyl group of this
side chain lies below and to the left of the nicotinamide behind Leu-203
(reproduced from Weinhold et al. [64], with permission of the corresponding author).
[14]
[15]
[16]
reactions which proceed via a bent transition state, suggesting that this particular enzymatic reaction has a linear
transition state.
[17]
[18]
Acknowledgements
[19]
[20]
References
[21]
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