Anomeric Specificity of the Alkaline Form of Fructose 1, BDiphosphatase from Rabbit Liver*

Abstract The preferred configuration of the active substrate for rabbit liver fructose 1,6-diphosphatase has been determined by techniques based on rapid quench kinetics to be the α anomer of fructose-1,6-P2. Utilization of the β anomer, however, is also catalyzed by the enzyme with a rate coefficient 5- to 10-fold less than that for the α anomer.

The anomeric distribution of fructose-l ,6-l'* in neutral aqueous solution previously has been shown by 1% NRlR to bc in a ratio of 4: 1 (/3:ol) (1) with essentially no acyclic (keto) form present ( _< 1 yO) (2, 3). In order to determine the anomeric substrate specificity of fructose 1 ,6-diphosphatase, there was added an amount of enzyme capable of reacting with all of the substrate of a particular anomeric configuration within a time span that is short with respect to the rate of anomerization.
Thus, if one anomer were reactive, one should observe its rapid conversion to fructose-6-l' followed by subsequent product formation that would be determined by the anomerization rate. This latter phase, ideally, should be independent of enzyme concentration if the rate-determining process is simply spontaneous or buffercatalyzed anomerization.
On the other hand, if both anomers can be utilized by the enzyme, i.e. the enzyme either catalyzes anomerization to the reactive configuration or lacks absolute anomeric specificity, the duration of both the initial and latter phases of product formation should depend on enzyme concentration.
This phenomenon has been observed previously for phosphoglucose isomerase (4).
The results of t,he rapid quench experiments are exhibited in terms of mole fraction fructose-6-P formed (ft) at various times. Under the preselected conditions where the initial ratios of [ET]:KM and [fi-fructose-l ,6-P2]:KM are 18 to 36 and 200, respectively, the true velocity is effectively that of V,,,,, (5). The kinetics remain zero order in substrate to greater than 80% hydrolysis for the @ anomer at all enzyme levels. Some justification for the assumption that KM for both (Y and fl anomers is identical is derived from earlier studies utilizing analogs including (Y-and P-methyl-n-fructofuranoside-1 , 6-PZ (6)  In Fig. 1 are displayed plots of ft versus time for two enzyme concentrations.
The graph is biphasic for the two experimental sets. An initial rapid forrnation of about 35% fructose-6-P is followed by a slower rate of product formation.
It is apparent that the slopes of the iuitial and second phases are dependent on enzyme concentration, both slopes doubliug (within experimental error) for a a-fold increase in fructose 1 ,6-diphosphatase. Extrapolation of the secoud phase to time zero yields in the two cases an St value of 0.2, signifying that 20% of fructose-l, 6-1'2 is more rapidly converted to product.
The agreement between extrapolated values at the two levels of enzyme supports the premise that the enzyme concentration is sufficiently high to reduce rapidly to a limiting negligible value the equilibrium conceutration of the form of fructose-l, 6-P, utilized by the enzyme. The observed percentage corresponds to preferred utilization of the 01 anomer of fructose-l, 6-P2.
Values for the slopes of the iuitial and second phases have been plotted as a function of enzyme concentration (Fig. 2). The lower line which describes only the conversion of fl anomer to product permits direct calculation of kg, the first order rate constant for decomposition of a presumed enzyme-/-fructose-l, 6-1'~ complex.
That the upper line contaius contributions for the conversion of both cy and p anomers to fructose-6-l' is indicated by the point of intersection of the two phases being >20% (Fig.  1). Insofar as the analog data are applicable, the initial phase may be solved for k a, the first order constant for decomposition of the enzyme-c-fructose-l, 6-P2 complex as follows. Assuming KMa N_ KMo, and since under our initial conditions [fructose-1,6-Pz] > E,, the initial distribution of the two complexes is 4 : 1 favoring the enzyme-fl-fructose-l , 6.P2 complex, provided binding is not rate-determining.
Consequeutly, 0.2 k, = k,, -0.8 kp where k,, is the observed first order rate constant for the initial phase.* The values for 12 u and ka determined according to this procedure are 0.07 i 0.02 and 0.009 + 0.002 (rnM. ml/unit .s), respectively.
Under the usual assay conditions, i.e. S, >> ET, the defined value for 1 unit of fructose 1,6-diphosphatase is 0.017 pmole 0; the value calculated from the above parameters, 0.021 + 0.005 pmole s-i, is in good agreement.
In essence the enzyme apparently may utilize the (Y anomer about 5 to 10 times more rapidly than the /3 anomer, although, as a consequence of the greater concentration of the p anomer in a  1 (lefl). Time course of fructose-6-P formation from (CX + 8) fructose-1,6-P*.
solution of fructose-l ,6-Pz, the rate of turnover of substrate arises from reaction of both anomers. Finally one may note the significance of these results as they bear on the mechanism of fructose 1,6-diphosphatase action. The fact that both initial and second phases are dependent on enzyme concentration argues against a scheme in which the enzyme interacts solely with one configuration of the sugar-1'2. If this were the case, the rates associated with the second phase would be independent of enzyme concentration.
Two main possibilities remain: (a) the enzyme catalyzes the hydrolysis of OLfructose-l ,6-P2 and conversion of fi to the reactive o( anomer or (b) the enzyme is relatively nonspecific and can act on both anomers.
A variation of (a) is to catalyze the ring opening of both a and p to the common keto intermediate prior to dephos-phorylatiom3 Rcgnrdless of the actual scheme, fructose I, 6-diphosphatase evidently is able to employ the greater thermodynamic instability of the cr anomcr for a rate advnritage.
Finally, it should be noted that these studies were performed with the alkaline form of fructose 1,6-diphosphatase.
Although the inhibition patterns for a series of substrate analogs with both the alkaline and neutral forms of the enzyme are identical (7), one camlot entirely discount the possibility that the anomeric specificity may be dcpcndcnt to some degree 011 the nature of the enzyme preparation employed.
ture by mixing equal volumes of enzyme and reactant solutions followed by rapid quenching with two volumes of 10% HClOd. Approximately 0.4 to 0.5 ml of quenched solution was collected. As a precaution against a systematic unrecognized error, such as enzyme denaturation, the reaction times were varied randomly during the course of an experiment with a given ET. Stock enzyme solutions varied from 35 to 85 units per ml of fructose 1,6diphosphatase in a diluent containing 0.1% bovine serum albumin, 0.5 mM MnClz, and 0.04 M glycine (pH 9.2). Enzyme in 5 x 1O-3 M malonate (pH -6.8) and 5 X 10m4 M fructose-l, 6-PZ (as eluted from a column) was concentrated via an Amicon ultrafiltration cell, repeatedly flushed with 0.04 M glycine (pH 9.2), treated with MnC& at 25", rechilled, and reflushed extensively with 0.04 M glycine. Enzymatic analysis showed the fructose 1,6-diphosphatase solution to be free of fructose phosphates. The composition of the stock reactant solution was 0.04 M glycine (pH 9.2), 0.5 mM MnC12, and 0.40 mM fructose-l ,6-Pz. The quenched reaction mixture, maintained at 0", was then centrifuged and the supernatant was removed and adjusted to pH 9.2 with 50% KOH.
Sample aliquots initially were analyzed for fructose-6-P by the standard coupled enzyme assay (10) and the unreacted fructose-l, 6-PZ then was measured by addition of fructose 1 ,6-diphosphatase.
The total recovered sugar phosphates were >90% that of the stock reactant regardless of enzyme concentration. MATERIALS AND METHODS

REFERENCES
Rabbit liver fructose 1,6-diphosphatase was purified according to published procedures (8,9). LIean specific activity unless otherwise specified was 15 units per mg. n-Fructose-1 ,~-Pz was obtained from Sigma. Rapid quench kinetic experiments utilized a Durrum Multi &Iixer as modified by the authors.