(Received for publication, June 21, 1995; and in revised form, July 27, 1995)
From the
Bacterial resistance to aminoglycoside-aminocyclitol antibiotics
is mediated primarily by covalent modification of the drugs by a
variety of enzymes. One such modifying enzyme, the 3`-aminoglycoside
phosphotransferase, which is produced by Gram-positive cocci such as Enterococcus and Streptococcus inactivates a broad
range of aminoglycosides by ATP-dependent phosphorylation of specific
hydroxyl residues on the antibiotics. Through the use of dead-end and
product inhibitor studies, we present the first detailed examination of
the kinetic mechanism for the 3`-aminoglycoside
phosphotransferase-IIIa. Initial velocity patterns deduced from
steady-state kinetics indicate a sequential mechanism with ordered
binding of ATP first followed by aminoglycoside. Dead-end inhibition by
AMP and adenylyl-imidodiphosphate is competitive versus ATP
and noncompetitive versus kanamycin A. Dead-end inhibition by
tobramycin, a kanamycin analogue lacking a 3`-OH, is competitive versus both kanamycin A and uncompetitive versus ATP,
indicative of ordered substrate binding where ATP must add prior to
aminoglycoside addition. Product inhibition by kanamycin phosphate is
noncompetitive versus ATP when kanamycin A is held at
subsaturating concentrations (K), whereas no
inhibition is observed when the concentration of kanamycin A is held at
10 K
. This is consistent
with kanamycin phosphate being the first product released followed by
ADP release. The patterns of inhibition observed support a mechanism
where ATP binding precedes aminoglycoside binding followed by a rapid
catalytic step. Product release proceeds in an ordered fashion where
kanamycin phosphate is released quickly followed by a slow release of
ADP. Aminoglycoside substrates, such as kanamycin A, show substrate
inhibition that is uncompetitive versus ATP. This indicates
binding of the aminoglycosides to the slowly dissociating (E
ADP) complex at high drug concentrations. These
experiments are consistent with a Theorell-Chance kinetic mechanism for
3`-aminoglycoside phosphotransferase-IIIa.
The development of antibiotic chemotherapy during the early part of the century has been paralleled by an increase in bacterial resistance to these drugs. Microorganisms have evolved that elude the cytotoxic effect of antibiotics by a variety of means including alteration of targets and chemical modification. The aminoglycoside-aminocyclitol antibiotics are one class of drugs subject to the latter mechanism of resistance. This family of antimicrobial agents includes streptomycin, gentamicin, and kanamycin A as well as many others (Fig. 1)(1) . The aminoglycosides can be grouped into three classes: 1) 4,6-disubstituted deoxystreptamine compounds such as kanamycin, 2) 4,5-disubstituted deoxystreptamine aminoglycosides such as paromomycin, and 3) those compounds without a deoxystreptamine ring, which include such drugs as streptomycin. They are used world wide but are subject to a broad spectrum of enzymatic inactivations. Aminoglycosides can be rendered inoffensive to the target bacteria by chemical modification catalyzed by a variety of enzymes including the O-adenyltransferases, the O-phosphotransferases, and the N-acetyltransferases(2) . The O-phosphotransferases are widely distributed in nature and are comprised of at least 20 different phosphotransferases depending upon the regiospecificity of hydroxyl group modification (for review, see (3) ). One particular phosphotransferase, 3`-aminoglycoside phosphotransferase-IIIa, produced by the opportunistic pathogens Enterococci(4) and Staphylococci(5) , regiospecifically modifies the 3` position of the 6-aminoglucose ring of kanamycin A and phosphorylates a variety of 4,5-disubstituted deoxystreptamine aminoglycosides as well(6) .
Figure 1: Structure of 4,5-disubstituted deoxystreptamine and 4,6-disubstituted deoxystreptamine aminoglycoside antibiotics.
To date, few aminoglycoside modifying enzymes have been subject to extensive kinetic or mechanistic studies. The 3-aminoglycoside acetyltransferase-I was demonstrated to follow a random kinetic mechanism (7) as was the bifunctional 6`-acetyltransferase 2"-phosphotransferase enzyme(8) . A third acetyltransferase (AAC(6`)-4) has been established to follow a rapid equilibrium random sequential kinetic mechanism(9) . The 2"-aminoglycoside nucleotidyltransferase was shown to inactivate the antibiotics through a Theorell-Chance kinetic mechanism (10) where the release of the nucleotidylated aminoglycoside is the rate-determining step in the reaction. The aminoglycoside phosphotransferases have not been subject to rigorous kinetic analysis with the exception of the 3`-aminoglycoside phosphotransferase-IIIa, where the groundwork has been laid by initial structure-function analysis(6) . We report herein, through the use of initial velocity studies, dead-end inhibitors, product inhibition patterns, and substrate inhibition, that 3`-aminoglycoside phosphotransferase-IIIa follows an ordered substrate addition and an ordered product release, which limits the rate of reaction and thus supports the occurrence of a Theorell-Chance mechanism.
where represents simple Michaelis-Menten kinetics and represents substrate inhibition kinetics.
Initial
velocities were fit to by nonlinear least squares where K = k
/k
and represents the
true dissociation constant of the first substrate in an ordered Bi Bi
system.
All inhibition data were fit by nonlinear least squares to as indicated in Table 1.
In all equations, v represents a measured velocity, V represents the maximal velocity,
[S] is the concentration of varied substrate, K
is the Michaelis-Menten constant, I is the
inhibitor concentration, K
is a slope inhibition
constant, K
is an intercept inhibition constant,
and K
is the true substrate inhibition constant.
Data were obtained in duplicate at high substrate concentrations and at least in triplicate for low substrate concentrations. Each data point was individually fitted to the above equations without further weighting. Errors given for the calculated parameters are standard errors for the best-fit to the equations by nonlinear regression(11) .
Figure 2:
Initial
velocity patterns for 3`-aminoglycoside phosphotransferase-IIIa.
Double-reciprocal plot at five concentrations of ATP. Fixed
concentrations of ATP were 5 µM (), 10 µM (
), 50 µM (
), 200 µM
(
), and 1000 µM (
). Data were fit to by Grafit.
Figure 3:
Uncompetitive inhibition of
3`-aminoglycoside phosphotransferase-IIIa through the use of the
dead-end inhibitor tobramycin. Double-reciprocal plot at six fixed
concentrations of tobramycin. Fixed concentrations of tobramycin were 0
µM (), 0.5 µM (
), 1.0 µM (
), 1.5 µM (
), 2.0 µM (
), and 2.5 µM (
). Data were fit to by Grafit. Inset, intercept replot for ATP versus [tobramycin].
AMP was found to be a
weak competitive inhibitor of ATP (Table 1; Fig. 4a) with a K of 4.9 ±
0.59 mM. It was also found to be a noncompetitive inhibitor of
kanamycin A with a K
of 10 ± 2.4 mM and a K
of 7.8 ± 0.51 mM (Table 1, Fig. 4b). This is consistent with an
ordered Bi Bi substrate addition where ATP is the obligate first
substrate followed by aminoglycoside addition.
Figure 4:
Inhibition of 3`-aminoglycoside
phosphotransferase-IIIa through the use of the dead-end inhibitor AMP. A, competitive inhibition of ATP. Double-reciprocal plot at
four fixed concentrations of AMP. The concentration of kanamycin A was
held at 100 µM (10 K
).
The concentrations of AMP were 0 mM (
), 2.5 mM (
), 5.0 mM (
), and 10.0 mM (
).
Data were fit to by Grafit. Inset, slope replot
for ATP versus [AMP]. B, noncompetitive
inhibition of kanamycin A. Double-reciprocal plot at four fixed
concentrations of AMP. The concentration of ATP was held at 25
µM. The concentrations of AMP were 0 mM (
),
2.5 mM (
), 5.0 mM (
), and 7.5 mM (
). Data were fit to by Grafit. Inseta, slope replot of kanamycin A versus [AMP]; inset b, intercept replot of kanamycin A versus [AMP].
A second nucleotide
analogue, AMP-PNP (a nonhydrolyzable ATP isostere) was found to be a
competitive inhibitor of ATP (Table 1) with a K of 350 ± 50 µM. It was also found to be a
noncompetitive inhibitor of amikacin with a K
of
990 ± 80 µM and a K
of 1.8
± 0.74 mM. These results are consistent with inhibition
results obtained using AMP and indicate an order of obligate ATP
addition first followed by aminoglycoside addition.
Figure 5:
Substrate inhibition of 3`-aminoglycoside
phosphotransferase-IIIa through nonproductive binding of
aminoglycosides. A, intercept replot versus [paromomycin]. The data were obtained by steady-state
kinetics for ATP at various concentrations of paromomycin (2-75 K). B, slope replot versus [paromomycin]; C, intercept replot versus [kanamycin A]. The data were obtained at
various fixed saturating concentrations of kanamycin A (10-100 K
).
Figure 6:
Inhibition of 3`-aminoglycoside
phosphotransferase-IIIa through the use of product kanamycin phosphate. A, noncompetitive inhibition of ATP by kanamycin phosphate.
Double reciprocal plot at four fixed concentrations of kanamycin
phosphate. The concentration of kanamycin A was held at 12.6
µM. The concentrations of kanamycin phosphate were 0
µM (), 100 µM (
), 300 µM (
), and 1000 µM (
). Data were fit to by Grafit. Inset a, slope replot of ATP versus [kanamycin phosphate]; inset b, intercept
replot of ATP versus [kanamycin phosphate]. B, no inhibition of ATP by kanamycin phosphate.
Double-reciprocal plot at four fixed concentration of kanamycin
phosphate. The concentration of kanamycin A was held at 126
µM. The concentrations of kanamycin phosphate were 0
mM (
), 0.5 mM (
), 1.0 mM (
), and 2 mM (
). Data were fit to Equations
4, 5, and 7 by Grafit. Inset a, slope replot of ATP versus [kanamycin phosphate]; inset b, intercept
replot of ATP versus [kanamycin
phosphate].
Determination of the kinetic mechanism of the aminoglycoside
detoxifying enzyme, 3`-aminoglycoside phosphotransferase-IIIa required
the combination of several techniques. Double-reciprocal plots of
initial velocity data have an intersecting pattern of lines indicative
of a sequential mechanism and excluding the possibility of a ping-pong
mechanism. This intersecting pattern of lines is observed for kanamycin
A as well as for paromomycin (4,5-disubstituted aminoglycoside) and
amikacin (which shows no substrate inhibition). This demonstrates that
the 3`-aminoglycoside phosphotransferase-IIIa follows a sequential
mechanism for all classes of aminoglycosides as well as for the
``poor'' substrate amikacin. A K of
26.0 ± 8.4 µM with kanamycin A as the fixed
substrate was calculated fitting data to . This value is in
agreement with a K
of 27.7 ±
3.7 µM reported previously(6) . This indicates
that K
= K
, where K
= k
/k
and K
= k
k
/k
(k
+ k
) and suggests that binding of ATP
is in rapid equilibrium(12) .
Substrate binding order was probed using substrate analogue dead-end inhibitors of both the nucleotide and aminoglycoside substrates. Tobramycin was found to be a potent competitive inhibitor of aminoglycosides and an uncompetitive inhibitor of ATP. The observation of uncompetitive inhibition in a sequential mechanism is diagnostic of an ordered mechanism where ATP binds prior to aminoglycoside binding (inhibitor binding)(14) . Both the nonhydrolyzable ATP analogue AMP-PNP and AMP were found to be competitive inhibitors of ATP and noncompetitive inhibitors of kanamycin A. These data support an ordered substrate addition where ATP binding is required prior to aminoglycoside binding.
Product
inhibition studies were initiated to elucidate the order of product
release. Given that ADP product release is coupled to pyruvate
kinase/lactate dehydrogenase for an assay of enzymatic activity, this
obviates the use of ADP as a product inhibitor. Kanamycin phosphate,
which we generated using purified 3`-aminoglycoside
phosphotransferase-IIIa, was therefore analyzed as a product inhibitor
of ATP. Initial studies involved holding kanamycin A at subsaturating
concentrations (K) while varying
ATP. A pattern of noncompetitive inhibition was observed with a K
of 10 ± 7.1 mM and a K
of 3.7 ± 0.47 mM. When
kanamycin A was held at saturating concentrations (10 K
), no inhibition was observed.
This pattern of inhibition is consistent with release of kanamycin
phosphate first followed by release of ADP. The observed elimination of
product inhibition by saturation with kanamycin A excludes a simple
ordered Bi Bi kinetic mechanism. It is consistent, however, with a
Theorell-Chance mechanism, which is a special case of the ordered Bi Bi
mechanism where the central complex (E
ATP
kan
&rlarr2; E
kan-phos
ADP) becomes kinetically
insignificant and does not contribute to the rate of reaction as
presented in Fig. SI(15) . This mechanism involves a
rapid release of kanamycin phosphate followed by a slower rate-limiting
release of ADP as the second product. The existence of the ternary
complex has been established by initial velocity studies, although its
existence is transient and kinetically insignificant, therefore the
product inhibition patterns are consistent with a ping-pong mechanism.
Figure SI: Scheme IKinetic mechanism and substrate inhibition of 3`-aminoglycoside phosphotransferase-IIIa.
Also compatible with the existence of a Theorell-Chance mechanism is
a 1.15-fold difference in k values and a
1.38-fold difference in k
/K
values
determined for ATP with several aminoglycoside fixed second substrates (Table 2). This marginal increase in both k
values and k
/K
values is
consistent with a Theorell-Chance mechanism, where the fixed substrate
is the second substrate in the binding order(13) . In a
double-reciprocal family of plots with ATP as the variable substrate,
all plots collapse into a single overlapping line. As presented in Fig. SI, changing the second substrate (i.e. aminoglycoside) would have very little effect upon the overall
rate of reaction, given that it is released prior to the proposed
rate-limiting segment of the mechanism. As the rate-limiting segment of
the reaction is dependent upon the nucleotide substrate, one would
expect a more significant k
and k
/K
effect
by using alternative fixed nucleotide substrates other than ATP (13) . This, however, is not possible when using the coupled
PK/LDH system.
Uncompetitive substrate inhibition (intercept linear)
by aminoglycosides versus ATP is indicative of nonproductive
binding of the aminoglycoside, at high concentrations, to the (EADP) enzyme form following kanamycin phosphate release (Fig. SI). The formation of this (E
ADP
I) complex (where I is the
aminoglycoside) must exist given that the nonproductive binding of the
aminoglycoside must be isolated from ATP addition by irreversible steps
(in order to observe uncompetitive inhibition)(16) . The
release of ADP from the (E
ADP) complex occurs more
slowly than the catalytic step and subsequent kanamycin phosphate
release. The slow release of ADP allows this form of the enzyme to
accumulate, and thus at high kanamycin substrate concentrations the
aminoglycoside is able to bind the (E
ADP) complex and
partially block ADP release from the (E
ADP
I)
complex.