The catalytic mechanism of flavin dependent C-N bond oxidations of amino acids can involve three
catalytic mechanisms. One mechanism involves initial carbanion formation through the abstraction of α- H+ by a catalytic base, followed by removal of H+ from the –NH3+ group either by an active-site residue
or solvent resulting in the rearrangement of electrons to reduce the flavin (Scheme 2.4 a-b-c). The mode
of transfer of reducing equivalents to the flavin cofactor after the initiation of carbanion is not clear,
although formation of N5 or C(4a) adducts with the flavin cofactor has been proposed 9, 43-46. The mech-
cleavage happening first followed by the adduct formation and the cleavage of NH leading to flavin re-
duction. The second mechanism involves nucleophilic attack of the lone pair of amine nitrogen at the
electrophilic C(4a) site of flavin forming an adduct with a concomitant abstraction of α-H+ by the N5 atom of the flavin as seen in Scheme 2.4, f-g-h. Electronic rearrangement results in the breaking of the
product imine from the reduced flavin14. For a protonated amino acid, its deprotonation is essential prior
to the attack at C(4a) atom of flavin. Therefore, this mechanism also requires for NH and CH bond cleav-
ages to occur in separate steps. The third mechanism involves cleavage of NH bond to deprotonate the
substrate amine thereby activating it for the transfer of a hydride directly from its α-C to the flavin cofactor
(Scheme 2.4 d-e). Thus, the order of events starts with the removal of H+ from the protonated amine
followed by CH bond cleavage. If the cleavages are rate limiting then use of substrate and solvent kinetic
isotope effects reporting on CH and NH bond cleavages can be exploited as useful probes to study the
catalytic mechanism of substrate oxidation by flavin dependent oxidases, oxygenases and dehydrogen-
ases.
DAAO
D-Amino acid oxidase, a paradigm for amine oxidation in D-amino acids, glycine and N-methyl-
ated amino acids catalyzing enzymes 9, 34, 47-51, formation of carbanion has been accepted to be obligatory
due to the oxygen dependent catalysis of HCl elimination from β-Cl-alanine along with the generation of
normal oxidation product, β-Cl-pyruvate 46. Despite studies using 5-deazaFAD in D-amino acid oxidase
demonstrating the transfer of α-hydrogen directly on to the flavin consistent with a hydride transfer mech- anism 52, mechanism involving carbanion was still recognized as being viable 9. Deuterium and tritium
kinetic isotope effects determined using D-alanine, D-serine and glycine suggested that the CH bond cleav-
age was fully rate limiting while the lack of solvent KIE was taken as being against concerted mechanism
53. This was consistent with a carbanion and a hydride transfer mechanism 53. However, with the availa-
bility of crystal structure of D-amino acid oxidase, the carbanion mechanism was rendered less credible
32
of 15N isotope effects suggested that rehybridization of substrate amine to imine was concomitant with
the cleavage of CH bond which is consistent with a hydride transfer mechanism but not carbanion mech-
anism 54.
DADH
Bacterial DADH has broad substrate specificity compared to DAAOs. However, the arrangement
of key residues proposed to be important for catalysis remains conserved among the two enzymes.41 Tyr53
and Tyr249 with ionizable side chains are positioned within 5 Å from the α-C while Tyr53 is ~ 4 Å from
the product imine which is the site of deprotonation during substrate amino acid oxidation. Mutagenic
studies on the two tyrosines ruled out any proposed role as active site base for these residues (Results
from Chapter 4 in this dissertation). The binding as well as catalysis is not significantly affected by the
replacement of Tyr to Phe at these sites. Tyr53 in PaDADH is equivalent in position to Tyr224 in
pkDAAO which has been proposed to be a potential base.34, 37 Due to lack of direct mechanistic evidence
and in the wake of the conclusions made in PaDADH, Tyr224 is less likely to be an active site base.
Furthermore, the fact that the residue is not found conserved in the yeast DAAO makes the argument less
Scheme 2.4 Schematic representation of carbanion mechanism (a-b-c), hydride transfer (d-e) and polar
nucleophilic attack (f-g-h) as catalyzed by amino acid oxidases and dehydrogenases. Scheme borrowed
from Chapter 4
For catalytically slower D-Leu as substrate, molecular dynamics (MD) simulations on PaDADH
showed that the binding free energy is much lower when a protonated (zwitterion) substrate is bound in
the active site compared to the unprotonated form. This observation suggested that the zwitterionic form
of the substrate is preferred for binding. Evidence in favor of this conclusion also comes from the Kd
measurements which shows pH dependence with a pKa of 9.6 with tight binding at pH below the measured
pKa. But at a pH above the pKa the Kd value increases as does the rate constant for flavin reduction sug-
gesting that for catalysis the protonated amino acid needs to be deprotonated. Use of deuterium substrate
34
asynchronous. These results are consistent with hydride transfer, carbanion or polar nucleophilic mecha-
nisms.39 Mutagenic and isotope effects on the tyrosine mutants have been instrumental in settling the
mechanism of D-amino acid oxidation as being most consistent with the transfer of a hydride ion from
substrate onto the flavin in PaDADH. The lack of a strong base and any detectable intermediates along
with concerted synchronous deuterium isotope effects in the tyrosine mutants played against the carban-
ion9 and polar nucleophilic mechanisms.8, 9 After the magnitude of mechanistic studies on DAAO, Pa-
DADH has surfaced as another example of enzymes involved in amino acid processing with mechanistic
and structural data favoring hydride transfer mechanism as the means of substrate oxidation.
LAAO
Mechanistic studies on LAAOs have lagged that of DAAOs and PaDADH. No direct mechanistic
study probing the catalytic mechanism has been carried out on LAAOs. Within this context, it is interest-
ing to note that LAAO from Calloselasma adamanteus has been shown to be reversibly inactivated at a
pH above neutrality or upon freezing to -20 oC. Based on available crystal structures, active site topologies
and in comparison with the mechanistic data available for enantiomerically opposite DAAO, hydride
transfer mechanism from substrate L-amino acid to flavin has been proposed.36, 37 Base catalyzed hydride
transfer has been proposed for crLAAO and His223 is proximal to the substrate amine to take up such a
role.37, 42 However, a carbanion mechanism also cannot be ruled out in this scenario. Structural studies on
crLAAO with bound inhibitors and substrates showed that His223 assumed different conformations
strengthening the argument of its role as an active site base55. However, mutagenic work on conserved
His223 from crLAAO showed that the activity of His223Ser, His223Ala and His223Asn is comparable
to the wild-type and the native crLAAO. Therefore, it seems likely that His223 may be important for
substrate binding55 but not as a catalytic base in the reductive half-reaction. Bacterial LAAO lacks an
amino acid at the position homologous to His223 in snake venom LAAOs. Hydride transfer mechanism
has been strongly favored due to the lack of a strong catalytic base in the vicinity of the amino acid
first step in the demethylation of methyl and dimethyl lysine by LSD1, a MAO enzyme has been shown
to proceed via a hydride transfer mechanism using QM/MM studies.56