(Received for publication, November 9, 1994; and in revised form, January 23, 1995)
From the
Examination of conserved motifs on the cloned subunits of the
deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser
(DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or
Asn reduced dGK and dAK activities to less than 0.2%, whereas
replacement of Arg-79 with Lys, either on both subunits in tandem
(R79K), or on the dGK subunit only (R79K:dGK), yielded active but
kinetically modified enzymes. These were partially purified, and their
kinetic and regulatory properties were analyzed. For dAK activity, the V of the R79K:dGK enzyme was increased 28-fold,
with no change in the limiting K
for
dAdo, but with a slightly reduced K
for
MgATP. The V/K efficiency ratio of dAK was also increased
29-fold, but that of dGK was decreased to 5-10% due to a 10-fold
increase in K
for dGuo and a reduced V
. Therefore, the R79K substitution seems to
have a greater effect on dGuo binding than on that of dAdo, but dGK
modification appears to produce a stimulatory conformational effect on
the opposite subunit, resembling the known unidirectional activation of
dAK by either dGuo or dGTP.
The four deoxynucleoside kinases of Lactobacillus
acidophilus R26 exhibit a level of specificity for their
respective deoxynucleoside substrates previously unknown among this
class of enzymes. Although the lactobacilli contain a thymidine kinase
(TK), ()an activity common to nearly all prokaryotes and
eukaryotes, and which resembles the Escherichia coli enzyme in
its regulation, (
)the other three activities are apparently
unique in terms of their nucleoside specificities, structural
organization, and regulation. In contrast with human and other
mammalian deoxycytidine kinases(1, 2, 3) ,
which actually phosphorylate dAdo and dGuo, as well as dCyd, at a
common site, these three activities are found in lactobacilli as two
heterodimers, dGK/dAK (deoxyguanosine kinase/deoxyadenosine kinase) or
dCK/dAK (deoxycytidine kinase/deoxyadenosine kinase), and separate
catalytic functions have recently been assigned to each
subunit(4) .
Each subunit's activity is selectively
controlled by feed-back inhibition from its respective dNTP end
product, which (mounting evidence strongly suggests) binds to both the
nucleoside and ATP sites as a bisubstrate
inhibitor(4, 5) . The other remarkable regulatory
aspect of these enzyme is the unidirectional activation of dAK, which
in its unactivated state has only about one-fifth the activity of its
associated dCK or dGK. However, when dCyd or dGuo, or their
triphosphate homologs, occupy the respective dCK or dGK subunits, the
turnover on dAK is increased up to 6-fold. (Curiously, dAdo or dATP
exert very little stimulatory effect on dCK or dGK, in the opposite
direction.) This effect is purely a turnover phenomenon; the K values for dAdo are unchanged by dCyd
or dGuo(6, 7) . Recent evidence suggests that this
heterotropic activation of dAK is due to conformational changes,
analogous to the classical taut-to-relaxed inter-conversion, induced
upon the respective binding of dCyd or dGuo (or their triphosphates) to
the dCK or dGK active site (4, 8) . Under these
circumstances, the dAK of either dimer becomes more susceptible to
limited protolysis, presumably by toggling into an open or relaxed
state, whereas selective proteolytic inactivation of dGK abolished this
effect(4) . In a related experiment, photoaffinity labeling of
the dGK active site with 8-azidoadenine permanently locks dAK into an
activated configuration, again revealing conformational communication
between the two subunits of the heterodimer(8) .
It is of
great interest to identify the structural basis for these unusual
regulatory phenomena as well as the sequences contributing to the
enzymes' active sites. Scanning for homology with structural
elements shown to be related to function in other kinases reveals that
dGK/dAK contains the glycine- and arginine-rich motifs associated with
the ATP- or GTP-binding sites of many nucleotide-binding proteins (9, 10) and which are also highly conserved in the
adenylate kinases (11) and herpesviral thymidine kinases (HSV
sites 1 and 5)(12, 13) . Another small conserved
motif, -DRH- (HSV-TK site 3), found between the G-loops and the
arginine-rich regions of herpesviral thymidine kinases(12) ,
may be analogous to the similarly located DRS motif of dGK/dAK.
Mutations 4-12 residues further on the HSV-1 TK map (around
HSV-TK site 4) produced increased K values for thymidine, leading Darby et al.(14) to suggest the region may be part of a
thymidine-binding site. Random oligonucleotide mutagenesis leading to
the replacement of a number of residues in the intervening region
between sites 3 and 4 has also produced mutants with both increased and
decreased K
and k
values for thymidine(15) , whereas a double amino acid
substitution within this region of varicella-zoster TK (which is
partially conserved relative to HSV TKs) resulted in reduced thymidine
and thymidylate kinase activities and larger dissociation constants for
these and analogous substrates(16) . Significantly, random
mutagenesis of HSV-1 TK produced no active enzymes with modifications
within the DRH motif, an indication of its possible importance, and
site-directed mutagenesis of the corresponding Asp-162 completely
inactivated the enzyme(17) , making it difficult to say whether
the mutation affected substrate binding or turnover, or if it produced
global disruption of structure. But, the essential character of Asp-162
and its proximity to site 4 would make it seem likely that sites 3 and
4 are both part of a larger nucleoside-binding or recognition domain
for HSV-TKs.
The homology between the DRS motif of dGK/dAK and the DRH of HSV-TKs, although very limited in size, suggested the possible importance of these residues in some aspect of nucleoside binding and/or catalysis in Lactobacillus dGK/dAK. Furthermore, since the regulatory events described appear to be mediated through deoxynucleoside sites, it seemed more desirable to probe potential deoxynucleoside sites initially, rather than the recognizable ATP-binding elements.
After the polymerization/ligation reaction was continued for 90 min at 37 °C, the reaction was stopped by adding Tris-EDTA buffer. Closed-circular heteroduplex recombinant pBlueScript KS(+) was extracted from agarose electrophoresis gel and used in the transformation of E. coli XL1-Blue by electroporation in a Gene Pulser (Bio-Rad), according to the manufacturer's protocol. Transformants were grown on LB plates containing ampicillin, and each instance of mutagenesis was confirmed by DNA sequencing, using the dideoxynucleotide termination method(22) .
Both UMCE and mutant Lactobacillus enzymes were expressed in E. coli at levels amounting to at least 3% of the total soluble protein in crude cell extracts, judging from the intensities of the 27-kDa bands resolved by 12% SDS-polyacrylamide gel electrophoresis (Fig. 1). The quantities of dAK/dGK peptides expressed by UMCE and mutants apparently are identical, and they remain unchanged after 16 h of incubation with good aeration at 37 °C, which suggests that there is no specific or selective degradation of the expressed foreign proteins by E. coli proteases. The sizes of all of the expressed dAK and dGK peptides appear to be identical at about 27 kDa, in close agreement with the wild-type L. acidophilus R26 dAK/dGK peptides(4) . Crude extracts of all mutants were screened for activity by the standard assay. After partial purification of the expressed cloned enzyme activities, the relative quantities and molecular sizes were unchanged.
Figure 1: SDS-polyacrylamide gel electrophoresis profiles of expressed UMCE and mutant enzymes in crude extracts. Lanes 1 and 9, molecular mass markers (Bio-Rad low range); lane 2, pBlueScript transformed cell control; lane 3, UMCE; lane 4, D78A; lane 5, D78E; lane 6, D68N; lane 7, R79K; lane 8, R79K:dGK. Expressed protein is indicated by the arrow.
All three types of Asp-78 mutations (D78A, D78E, and D78N, involving both subunits) virtually eliminated both dAK and dGK activities, being less than 0.2% of the activities found in comparable UMCE fractions. As the activities of these mutants were deemed to be too low for reliable kinetics, further analysis was not attempted. While it is quite likely that Asp-78 is directly essential for the activities of each subunits, indirect effects such as disruption of folding cannot yet be ruled out as an explanation for the loss of activities.
Charge-conservative mutations at Arg-79, on the other hand, had a much more remarkable effect. While preliminary assays (not shown) revealed a reduction in the dGK activities of both the R79K and R79K:dGK mutants, the dAK activities were increased dramatically, whether or not the dAK subunit was altered. However, there was no apparent increase in the amount of peptide expressed. Preliminary enzyme activation and inhibition studies were carried out to determine whether these regulatory mechanisms might have been altered by R79K mutations. While both mutant enzymes remain susceptible to inhibition by end products dGTP or dATP, activation of dAK by dGuo is nearly eliminated in the case of the R79K:dGK mutant, while for the tandemly mutated R79K, there was even a 30% inhibition of the dAK activity upon addition of dGuo. In the opposite direction, dAdo produces, at most, a 10% stimulation of UMCE dGK, and this small stimulation is replaced by a comparable inhibition of the R79K:dGK mutant.
Effects of Arg-79 Mutations on Steady State
Kinetics-Because mutations at or near the active site of an
enzyme may alter its K values, or even its kinetic
mechanism, it is necessary to compare limiting K
and V
values (i.e. extrapolating
to saturation by both substrates rather than the apparent values
obtained by varying only one substrate) lest variable second-substrate
saturation produces apparent differences in reaction velocities.
Therefore, these parameters were determined for UMCE and each of the
Arg-79 mutants by steady-state kinetics in which both deoxynucleoside
and MgATP concentrations were varied (Table 2). The limiting K
for dAdo was increased severalfold in the tandem
mutant (7.5 µM in R79K) but was unchanged by the dGK-only
mutation. A greater effect is seen upon the binding of dGuo, with the K
for dGuo increasing about an order of magnitude
on both the single and tandem mutants.
An interesting contrast in
the effect of mutation on the K values for MgATP
occurs between the two subunits. Whereas both the single and tandem
mutations resulted in decreased K
values for MgATP
on the dAK subunit, i.e. whether or not the dAK subunit was
altered, the K
for MgATP on dGK was doubled for
both mutants. No change was produced in the K
for
MgATP on dAK upon the binding of dGuo to dGK of UMCE (data not shown).
The other conclusion which can be inferred from
these data is that, in terms of the V/K
``efficiency
ratios,'' the R29K mutations can be said to have had opposite
effects on the activities of the two subunits. Whereas the efficiency
of dAdo binding and turnover combined increased from 3- to 29-fold,
that of dGuo was decreased 90% or more. Since limiting values, i.e. real V
values and K
values, make up these ratios, it is even more apparent that
asymmetry exists between the two subunits, despite their identical
sequences within the motif which was mutagenized.
Far more compelling evidence for
unchanged kinetic mechanisms is provided by end product inhibition
studies. Earlier work from this laboratory has established that with
these enzymes, as well as with the dCyd kinase/dAdo kinase pair from L. acidophilus R26, the respective dNTP end products mimic the
effects of multisubstrate analogs(5) . Thus, in the case of
random substrate binding, the multisubstrate analog or the homologous
dNTP should compete with either substrate for the free enzyme, while
for an ordered kinetic path these compounds will compete only with the
leading substrate. For the wild-type dGK, dGTP competes with both dGuo
and MgATP, consistent with a random mechanism, while for dAK dATP
competes only with MgATP, the leading substrate in an ordered path, but
is noncompetitive toward dAdo (5) . Identical conclusions as to
differing kinetic mechanisms for the two activities were drawn earlier
from classical product-inhibition analyses. However, the end product
inhibition experiments used in the present work are practically simpler
than the classical product-inhibition experiments used to establish the
kinetic mechanisms of the wild-type enzymes, but provide clear-cut
results. Dixon plots (1/V versus [dNTP] at several
substrate concentrations) were used in obtaining numerical values for
the inhibitor dissociation constants(23) . The K values for end product inhibition against each substrate were
decreased up to 75% in dAK, but were increased about 2-fold in dGK by
both the R79K and R79K:dGK mutations (Table 3). However, neither
the dNTP inhibition pattern nor, therefore, the kinetic mechanism of
either enzyme was affected by mutations at Arg-79; the kinetic path for
dAK remains ordered, while that for dGK remains random. Therefore,
mutation at Arg-79 affects only the substrate binding and turnover of
the paired enzymes, not their kinetic mechanisms.
The heterodimeric deoxynucleoside kinases of Lactobacillus present an array of important fundamental questions relating to the control of DNA precursor biosynthesis. These include the mechanism of end product inhibition of each activity, the structural basis of the exquisite deoxynucleoside specificity exhibited by each catalytic subunit and the positive heterotropic stimulation of dAdo turnover by dCyd or dGuo, respectively.
We elected to probe first the dGK/dAK DRS motifs, the conserved elements which by analogy with HSV TKs seemed likely to be associated with the deoxynucleoside sites. While we have no details yet of the dGK/dAK heterodimer's tertiary structure, a very preliminary prediction of the protein's secondary structure by the method of Chu and Fasman (28) or Garnier, et al.(29) suggests that the -DRS- motif may be at the turn of a loop. Therefore, if this local structure is involved in the binding of substrates or the stabilization of intermediates in the enzyme reaction, most substitutions should cause dramatic effects on enzyme activities. Accordingly, site-specific replacement of Asp-78 of both subunits with Glu, Asn, or Ala virtually abolished both the dGK and dAK activities of the mutants.
From the
standpoint of uncovering the subtleties of enzyme regulation by subunit
interaction, a much more interesting site of mutagenesis is found at
Arg-79 of the Lactobacillus enzyme upon charge-conservative
replacements with Lys. Whereas the dGK activities of R79K and R79K:dGK
were reduced substantially, the dAK activities of those mutant enzymes
were elevated dramatically above that of UMCE. This phenomenon is
thought to be analogous to the opposite-substrate activation effect
reported previously with wild-type dCK/dAK and dGK/dAK (6, 7) in which up to 6-fold stimulation of dAK
activity occurs upon adding the opposite substrate, dCyd or dGuo,
respectively, to the dAK reaction mixture. Arg-79-Lys mutations of the
dGK subunit, either singly or in tandem with modification of the dAK
subunit, essentially abolish this stimulation by dGuo, but, at the same
time, produce an 8-28-fold increase in the V of dAK. Affinity-labeling of the dGK active site with
8-azidoadenine produced a comparable, if smaller, effect(8) .
We may presume that these modifications at or near what appears to be
part of the dGK active site induce a conformational change in the dGK
subunit similar to that produced by dGuo binding, and which, in turn,
toggles the dAK subunit into its activated conformation. The smaller
8-fold stimulation in the case of the tandem mutant is probably due to
some offsetting reduction of efficiency of the dAK subunit upon its
mutation. It is important to note, however, that for R79K:dGK the K
for dAdo is unchanged, whereas it increases
severalfold in the R79K mutant where both subunits are altered.
Stimulation by dGuo has been shown to be purely a V
effect, producing no change in the K
for
dAdo(7) , and the R79K:dGK mutation seems to have had an
identical effect. Recent work on the wild-type dGK/dAK in this
laboratory suggests that selective modification of the dGK subunit by
photoaffinity labeling may also lock dAK into its activated
configuration(8) , an effect which is mediated therefore
through the dGK subunit.
The R79K mutation has opposite effects on
the K values for MgATP at the two active sites,
but lower K
values on dAK would not account for
the stimulation as the assay is carried out under near-saturating
conditions with respect to MgATP. Since these mutations are not within
either of the putative ATP-binding domains of the protein, we
tentatively assume that global folding or interactions between domains
are affected. On dGK the K
for MgATP is already
much larger than on dAK, and is doubled again by the Arg-79 mutation.
It should be noted that, even for the wild-type enzyme, the K
for MgATP on the dGK subunit (1.7 mM)
is an order larger than on dAK (0.11 mM). Although the
conserved ATP-binding sites at the N termini are genetically identical,
they do not appear to be processed alike after translation in either Lactobacillus or the E. coli host(24) .
The evidence pointing to asymmetry between the tertiary and/or
quaternary structures of the two subunits can be summarized as follows.
(i) We note that they follow different kinetic pathways, one ordered
and one random, and the activation of dAK by dGuo or by the R79K:dGK
mutation does not alter this. However, the K for
dATP is decreased by about the same extent as the K
for MgATP, the leading substrate in the ordered mechanism. This
is consistent with the understanding that dATP behaves as a
multisubstrate analog bridging the deoxynucleoside and ATP-phosphate
sites. (ii) The one-way stimulation of the dAK activity by dGuo or
dGTP, by affinity labeling of the dGK subunit (8) and by R79K
mutagenesis also supports this notion. (iii) The opposite effects of
R79K replacement on the binding of MgATP to the two subunits, despite
similar sequences, also suggests differences in folding or contact
points of the heterodimer. For example, the subunits could be arranged
in a head-to-tail fashion, effectively burying one of the N termini.
The detailed mechanism of the enhancement of dAK activity by R79K
replacement is still unknown, but it seems most likely that Arg-79 is
located near the contact area, possibly in a loop, between the dAK and
dGK subunit, and the amino acid residues involved in the substrate
binding site of one subunit might effect on the overall rate-limiting
reaction steps of the paired subunit. Since the K for dAdo is not changed by the mutation on dGK, turnover of dAK
rather than substrate-binding is affected, and we may regard dGuo as a
positive heterotropic effector of a rate-limiting step in dAK
catalysis.