THE Joum,.~ OF BIOLOGICAL CHEMISTRY Vol. 253, No 7. Iseue of April 10, pp. 2093-2097, 1978

Printed m V.S.A

Selective Chemical Modification and lgF NMR in the Assignment of a pK Value to the Active Site Lysyl Residue in Aspartate Transaminase*

(Received for publication, August 22, 1977)

JUAN C. SLEBES AND MARINO MARTINEZ-CARRION§

From the Department of Chemistry, University of Notre Dame, Notre Dame, Indiana 46556

The simultaneous application of chemical modification of primary amines and the insertion of a lgF probe have been used to detect the nature and physical properties of the amino group influencing a selected region of the active center of the enzyme aspartate aminotransferase. The en- zyme, carbamylated at the NH,-terminal alanine (transam- inase’, produced from cyanate-treated holoenzyme) or at the active site Lys-258 (transaminase*, inactivated by car- bamylation of Lys-258 with KNCO), can accept phosphopyr- idoxyl trifluoroethylamine instead of the constitutive coen- zyme pyridoxal phosphate. The spin-lattice relaxation time, T,, of the fluorine atoms in the enzyme-bound fluorinate compound is 0.12 s and the spin-spin relaxation time, TO, is 6.4 ms. Both T, and T, values remain constant after carba- mylation of Lys-258. Assuming a simple treatment of the dipolar relaxation mechanisms as being due to IH-lYF inter- actions, a molecular rotation correlation time TV value of -80 ns is calculated for native and carbamylated enzyme. The complexes of phosphopyridoxyl trifluoroethylamine and transaminase’ or transaminase*, although catalytically inactive, retain the same affinity as those with native transaminase for the competitive inhibitors succinic acid and acetate anion.

Increasing ionic strength results in increasing values for the observed pK of Lys-258 determined from a pH depend- ence of the rate of enzymatic inactivation by KNCO treat- ment. The ionic strength effects are identical with those previously observed in the pK value of the protein group perturbing the ‘“F NMR of bound phosphopyridoxyl trifluo- roethylamine (Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio A. M., (1976) J. Biol. Chem. 251, 1853- 1858). The pH-dependent chemical shift changes of the single resonance of the fluorine probe introduced to native transaminase or transaminase’ appear to be due to the ionization of a single protein residue with a pK of 8.2. On

* This work was supported by Research Grants HL-11448 and GM- 20727 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department0 de Bioquimica, Facultad de Med- icine, Universidad de Chile, Santiago, Chile.

5 Research Career Development Awardee from the National Insti- tute of General Medical Sciences. To whom correspondence may be addressed. Present address, Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. 23298.

the other hand, the complex of phosphopyridoxyl trifluoro- ethylamine with apoenzyme* shows no pH effect on the chemical shift or line width of the l”F resonance. The evidence suggests that the NH,-terminal alanine does not exert influence on the environment of the active site lYF probe and that the observed effects on the NMR signal of this probe can be assigned to the ability of Lys-258 to ionize.

In order to inquire into the relationships of protein structure to function, natural or synthetic spectroscopic probes can be used. These probes report the properties of their local environ- ments. However, if two approaches, chemical modification of specific protein residues and spectroscopic probes, are used simultaneously, more specific answers about the nature and properties of the protein residues affecting the probe should be extractable.

The active site of supernatant glutamate-aspartate amino- transferase (EC 2.6.1.1.) has not been characterized fully. Previous work (1, 2) has implicated the lysine residue to which the coenzyme, pyridoxal-P’ is bound, as essential in the mechanism. Sulfhydryl groups may be essential for mainte- nance of enzyme structure (2-4) and aromatic residues could participate in coenzyme binding (5-7). Martinez-Carrion et al. (8) have shown that histidyl residues are destroyed by pho- tooxidation in the presence of methylene blue, with concomi- tant loss of activity and a histidyl residue being critical. Yet, in this enzyme, as in most well characterized proteins, very little is known about the properties of individual amino acid residues. Because of their possible functional implications, we have initiated a series of experiments involving the develop- ment of tools to inquire into the properties of active site amino acid residues. We have employed both insertion of sensitive physical probes (9) and selective chemical modification (10) of the topographic region(s) under scrutiny. Recently, we have reported (10) that in aspartate transaminase, if the conditions are properly chosen, the cyanate-sensitive e-amino group of Lys-258 or NH,-terminal (alanine) (or both) can be selectively blocked with cyanate. The holoenzyme, when labeled (one cyanate group/subunit) with KNCO at the NH,-terminal,

1 The abbreviations used are: pyridoxal-P, pyridoxal phosphate; pyridoxamine-P, pyridoxamine phosphate; apotransaminase’, apoenzyme produced from cyanate-treated holoenzyme; and apo- transaminase*, apotransaminase’ inactivated by carbamylation of Lys-258 with KNCO.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

very slow rate of inactivation in concert with a loss of activity. On the other hand, the cyanate-induced inactivation of apoas-

2094 Carbamylation and lgF NMR of Active Site pK Determinations

retains its original enzymatic activity. By contrast, the same treatment of the apoenzyme leads to an enzyme which has virtually no catalytic activity (10). This value is very similar to the pK, of 8.3 detected for a group influencing the chemical shift of a ‘“F-labeled probe inserted at the active site (9). Yet it is still not clear which amine is the perturber of the ‘“F NMR probe. Lys-258, the NH,-terminal alanine, or the amine in pyridoxyl trifluoroethylamine itself are the most likely candi- dates.

partate transaminase alone followed a pseudo-first order be- havior with a rate constant of 0.012 min’. Higher concentra- tion of cyanate (0.2 M KNCO) produced rapid and total inactivation with a pseudo-first order rate constant of 0.08 min-I. Under the same experimental conditions, no activity losses were detected when KNCO was replaced in the incuba- tion system by the same concentration of KCl.

In this paper, we show how chemical modification studies can help in the clarification of assignments of the amine groups affecting a ‘“F spectroscopic probe in the active site region.

EXPERIMENTAL PROCEDURES

Materials - Perfluorosuccinic acid and trifluoroacetic acid were from Pierce Chemical Co. 2,2,2-Trifluoroethylamine was purchased from PCR Inc. Alkaline phosphatase from Escherichia coli, malate dehydrogenase, NADH, pyridoxal-P, L-aspartic acid, and a-ketoglu- taric acid were acquired from Sigma and potassium cyanate from J. T. Baker Chemical Co. Phosphopyridoxyl trifluoroethylamine was prepared as described by Martinez-Carrion et al. (9).

Aspartate Transaminase- Supernatant aspartate aminotrans- ferase (EC 2.6.1.1) was isolated from pig heart in the pyridoxal-P holoenzyme form and apoenzyme, apoenzyme’, or apoenzyme* were prepared as previously described (6, 10). Protein concentrations were calculated from the optical densities at 280 nm where the extinction coefficient is 140,000 for a molecular weight of 94,000 (11).

Activity Determinations- Aspartate transaminase activity was measured spectrophotometrically by following the NADH utilization at 340 nm in a coupled assay containing in a total volume of 1 ml: excess malate dehydrogenase, 0.2 mM NADH, 6 mM a-ketoglutarate, 100 mM L-aspartate, and 50 mM sodium phosphate buffer (pH 7.5). One unit of activity was defined as the number of micromoles of NADH oxidized/min/mg of protein.

Cyanate Modification- The cyanate inactivation was studied by adding KNCO to enzyme solutions in the experimental conditions described in legends to figures and using the procedure previously reported (IO).

Nuclear Magnetic Resonance Measurements - ‘$F NMR chemical shifts were recorded on a Varian model XL-100 high resolution spectrometer at 94.1 MHz operating in pulsed Fourier transform mode as previously described (9). The titration curves were fitted with a Wang 700 B advanced programing calculator and the pH was controlled either by addition of 1 mM HCl or NaOH using a glass combination electrode and a Radiometer model 26 pH meter. Spin- lattice relaxation times, T,, were calculated using the progressive saturation technique. The values of spin-spin relaxation times, T,, were obtained from line width measurements and the relationship T, = l/(rAv). All samples contained 5 mM EDTA.

Ligand Binding to Carbamylated Transaminase- For the deter- mination of the dissociation constants, phosphopyridoxyl trifluoro- ethylamine. apoenzyme* complex was prepared using the carbamy- lated apoenzyme (apoenzyme’) as described previously (9, 10). Deionized enzymes were prepared by passage through mixed bed resins after extensive dialysis against dionized water and concen- trated by vacuum dialysis. The enzyme preparations, in a volume of 3 ml (50 mg/ml), were titrated either with sodium trifluoroacetate or perfluorosuccinate at constant pH using a 5-mm (outer diameter of a NMR tube) insert containing the acidic form of the ligand as external reference (12).

Effect of Zonk Strength on pH Dependence of the Rate Constant of Inactivation-The pseudo-first order rate of inac- tivation of aspartate transaminase by cyanate is due to carbamylation of the e-amino group of Lys-258 residue (10). The reaction is pH-dependent. In addition, we have suggested that observations of the chemical shift changes of the ‘$F NMR resonance of an active site ‘“F probe, phosphopyridoxyl trifluo- roethylamine are due to the ionization of a group in the protein near the 19F probe (9). The pK, of the detectable single ionization is markedly affected by ionic strength and those results were consistent with the effect of the ionization of an E- amino group whose pK value is lowered by electrostatic interactions of other positive charges (9). By the application of a kinetic technique (9, 13, 14) previously used in the carba- mvlation of asnartate aminotransferase at a fixed ionic strength and pH value, we determined the pseudo-first order rate constants of inactivation of the enzyme by KNCO. The rates of inactivation by 0.1 M KNCO were followed at 37” at several pH values and at varying ionic strengths. Plots of these rate constants versus pH had sigmoidal shapes with maximal rates below pH 8.0 (Fig. 2). From the observed pseudo-first order rate constants, (Iz,bs) values the cyanate concentration (C,), the dissociation constant of cyanic acid (K,), and the hydrogen ion concentrations (H+) plots of C&,,, versus j&/H+ (9) at different ionic strengths show the effect of ionic strength on the observed pK of the Lys-258 residue. The pK values obtained under these conditions are included in Table I. The marked dependence of these values on the ionic strength of the medium is in good agreement with similar observations using the ‘“F NMR signal of the active site-bound phosphopyridoxyl trifluoroethylamine probe (9).

Complex of Phosphopyridoxyl Trifluoroethylamine with Different Forms of Apotransaminase - The fluorinated pyri- doxamine-P derivative can combine stoichiometrically with either apoenzyme’ or apotransaminase* as efficiently as with noncarbamylated apoaspartate transaminase. The formation of the carbamylated apoenzyme . coenzyme derivative complex and detection of the stoichiometry of the reaction can be followed as in native apoenzyme by fluorescence, circular

RESULTS

Znactivation of Cytoplasmic Aminotransferase by Cyanate - The time course of inactivation of aspartate transaminase by 0.03 or 0.2 M KNCO at pH 7.4 and 37” is shown in Fig. 1. The susceptibility to inactivation depends, as reported earlier, in the presence or absence of coenzyme (10). Under these condi- tions, holoenzyme, which has the active site lysyl residue blocked by pyridoxyl-P, is functionally unaffected by the cyanate treatment. Phosphopyridoxyl trifluoroethylamine, which binds at the active site of apotransaminase shows a

FIG. 1. Effect of carbamylation on the activity of different forms of aspartate aminotransferase (5 X lo-” M): 1, holoenzyme; 2, phosphopyridoxal trifluoroethylamine apoenzyme complex; 3 and 4, apoenzyme incubated at 37” with 0.03 (lines 1, 2, and 3) or 0.2 M KNCO (line 4) in 0.05 M sodium phosphate buffer, pH 7.4.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

Carbamylation and ‘SF NMR of Active Site pK Determinations

FIG. 2. Left, pseudo-first order rate constants of inactivation of apotransaminase (5.0 x 1O-5 M) at 37” with 0.1 M KNCO in 0.05 boric acid/borax buffer at varying ionic strengths as a function of pH. Solid lines represent theoretical titration curves for a single ioniza- tion group. Right, plots of C,llz,b, ver.sU.s K,/(H+) from the data obtained in left graph as described in Refs. 9 and 14.

TABLE I Effect of ionic strength on the pK of a group at the active site of

aspartate transaminase determined by cyanate inactivation

Apoenzyme (4.9 x 10m5 M) was incubated with KNCO at different pH values varying the ionic strength with KCl. The conditions and procedure are described in the legend to Fig. 2.

Ionic strength VK 0.12 7.88 0.22 8.14 0.45 8.29 1.12 8.47

dichroism, and absorption spectral measurements (9, 15). The addition of 1 eq of the fluorinated compoundleq of apoenzyme’ results in inactivation of the apoenzyme’. The presence of an excess of pyridoxyl-P or pyridoxamine-P fails to displace the phosphopyridoxyl trifluoroethylamine as no recovery of activ- ity was obtained. However, as for unmodified apoenzyme (91, the apotransaminase’, which is carbamylated at the NH,- terminal alanine residue, can be regenerated after incubation of the apoenzyme’ . fluorinated coenzyme complexes with 1 M

phosphate buffer at pH 5; the regenerated apoenzyme’ can regain full enzymatic activity (data not shown) upon dialysis of the free fluorinated pyridoxamine-P derivative and addition of pyridoxal-P.

lgF NMR of Phosphopyridoxyl Trifluoroethylamine . Apoenzyme, Apoenzyme’, or Apoenzyme” Complex- The “l? NMR spectrum of phosphopyridoxyl trifluoroethylamine bound at the active site of apoenzyme’ consists of one reso- nance signal which, as for native apoenzyme . fluorinated derivative complex, is pa-dependent. The chemical shift changes with pH correspond to the titration of a single ionizing group in the range of 6.4 and 9.5 with a pK value of 8.45 k 0.08 (Fig. 3). On the other hand, no chemical shift was observed when phosphopyridoxyl trifluoroethylamine . apoen- zyme* complex (97% inactive) was used. Thus, the loss of pH dependence of the chemical shift occurs in the same enzyme preparation which lost activity as a result of a pH-dependent carbamylation of Lys-258 and not in those enzyme prepara- tions with carbamylated NH,-terminal residues.

19F NMR of Fluorinated Compounds - Fig. 4 shows the pH dependence of the chemical shift resonance in ‘“F NMR

FIG. 3. pH dependence of the chemical shift resonance (AS), in Hz, of 19F in phosphopyridoxyl trifluoroethylamine bound to the active site of 1 x 10m3 M aspartate transaminase apoenzyme in 0.1 M KC1 and 1 PM EDTA. 0, apotransaminase*; 0, apotransaminase’; A, native apotransaminase. Solid line with a 150 Hz amplitude is a theoretical titration curve for a single ionization of a group with a pK of 8.45 + 0.08.

100’ 2 4 6 0

-4 a 1 I

0

-2 4 6 8 IO

PH

FIG. 4. pH dependence of the chemical shift resonance of lYF in fluorinated compounds in the absence of enzymes. Top, 2,2,2-trifluo- roethylamine (100 mM). Solid line, a theoretical titration curve of a simple ionization group with a pK of 5.65. Bottom: 0, phosphopyri- doxyl trifluoroethylamine (10 mM); 0, the coenzyme derivative after alkaline phosphatase treatment; -, theoretical titration curves for a single ionization with a pK of 5.90 and 5.50, respectively.

titration of the 2,2,2 trifluoroethylamine and the phosphopyr- idoxyl derivative of this compound. The pK values for these dissociations were 5.6 and 5.9, respectively.

The pK of the secondary amine in the pyridoxyl derivatives of trifluoroethylamine is dependent on the phosphorylation of the pyridoxyl moiety. The higher pK value for the phospho- pyridoxyl trifluoroethylamine is probably due to the influence of the phosphate group. Incubation of the phosphorylated

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

Carbamylation and lsF NMR of Active Site pK Determinations

derivative with 1 unit of alkaline phosphatase for 16 h at 25 results shown in Table II indicate that both inhibitors bind to led to complete hydrolysis (monitored by 31P NMR (16)) of the the enzyme carbamylated at the Lys-258 residue as well as to 5’-phosphate and a decrease of the pK value to 5.5 (Fig. 4). native transaminase.

19F Relaxation Times of Enzyme-bound Phosphopyridoxal Trifluoroethylamine - Interactions between the protons of a protein and associated fluorine nuclei are responsible for a major fraction of the relaxation rate exhibited by the fluorine nuclei (17, 18). In phosphopyridoxyl trifluoroethylamine, the protons most likely to exert dipole-dipole relaxation over lSF nuclei are the methylene protons or other protons in the protein which are in proximity due to the secondary or tertiary structure of the macromolecule. The interactions could be altered by the presence of substrates or inhibitors which either bind directly to parts of the fluorinated coenzyme analogue or perturb some vicinal protons in the protein structure. Thus, calculation of T, and T, could help in detecting some of the ligand changes in the immediate vicinity of the CF, probe. The measurements can also help in calculating 7, (171, the correlation time, which represents isotropic tumbling of the protein molecule as a whole. The ratio T,/T, ought to be large for isotropic motion and any changes of these dynamic param- eters due to presence of enzymatic ligands can in principle be detectable.

DISCUSSION

Values of T, = 0.12 * 0.02 s and T, = 6.4 ms were obtained for the enzyme alone or in the presence of 50 mM succinate or 0.1 M aspartate. These values did not change with varying pH in the range 5.5 to 8.0. Carbamylation of Lys-258 (carbamyla- tion of apoenzyme’) did not alter these results. Calculation of 7, from the TJT, ratio according to the procedure of Hull and Sykes (17) produces a 7, value of 80 ns. Phosphopyridoxyl trifluoroethylamine alone has a T, relaxation time of 1.6 s which is also independent of pH from pH 5 to 8.

Effect of Active Site Ligands on Phosphopyridoxyl Trifluo- roethylamine .Apoenzyme or Apoenzyme* Complex- After ac- tive site chemical modifications, it is difficult to test for the inactive enzyme’s ability to bind substrates. Fortunately, in this enzyme, binding of inhibitors can be monitored even in chemically modified preparations (9, 12). Thus, it is feasible to investigate for the possible participation of Lys-258 on ligand binding if the carbamylated enzyme in holoenzyme* (fluori- nated pyridoxyl derivative at the active site) is tested for its ligand binding ability.

Cyanate treatment of aspartate transaminase does not lead to inactivation of the enzyme if the coenzyme is present (10). This is also true if phosphopyridoxyl trifluoroethylamine is at the active site instead of pyridoxal-P. The fluorinated com- pound protects carbamylation of the active site Lys-258 most likely through a steric hindrance mechanism as there is no evidence for a conformational change induced by the binding of this coenzyme analogue to the apoenzyme (9). In holo- transaminase, KNCO carbamylates alanine (NH,-terminal residue) and some nonessential lysyl residues producing holo- transaminase’. I f apotransaminase reacts with KNCO, then Lys-258 is carbamylated producing apoenzyme*. Both apotransaminase’ (active) and apotransaminase* (inactive) do bind pyridoxal-P (10) and also bind the fluorinated analogue phosphopyridoxyl trifluoroethylamine. The resulting holoen- zyme is inactive due to the presence of the catalytically unproductive fluorinated coenzyme. Even though the complex is very stable, apoenzyme’ or apoenzyme* can be regenerated at will.

To correlate spectroscopic data obtained by a probe intro- duced into a chemically modified protein and into a native enzyme, one must show that the probe binds identically in both forms. We have previously reported that the fluorinated analogue resembles pyridoxamine-P rather than pyridoxal-P (9). After carbamylation of the NH,-terminal alanine or Lys- 258, the fluorinated transaminase’ or transaminase* also binds anions or inhibitors as the pyridoxamine-P form does in native enzyme. These findings, as well as the degree of fluorescence quenching and extent of positive dichroicity due to bound fluorinated coenzyme, strongly suggest a fit of the phosphopyridoxyl trifluoroethylamine in apoenzyme’ or apoenzyme* similar to that in native apoenzyme.

The affinity for sodium trifluoroacetate and perfluorosuccin- ate was measured in the holotransaminase* with carbamy- lated Lys-258 as well as in unmodified holotransaminase. The

TABLE II

Ligand affinity of native and carbamylated phosphopyridoxyl trifluoroethylamine holotransaminase

The dissociation constants were obtained plotting the reciprocal of the observed chemical shift of the methylene fluorines of perfluo- rosuccinate and the methyl fluorines of trifluoroacetate at varying concentrations of these ligands in the presence of a constant concen- tration of the phosphopyridoxyl trifluoroethylamine.apoenzyme complex (1.8 x 10m3 M) at 30”, pH 8.0 (9, 12). Carbamylated phospho- pyridoxyl trifluoroethylamine holotransaminase is apotrans- aminase* with phosphopyridoxyl trifluoroethylamine stoichiometri- tally bound at the active site.

Enzyme form (&I Ligand Native fluorinated Carbamylated and

enzyme fluorinated enzyme ?TlM

The kinetics of KNCO inactivation of the apoenzyme’ are consistent with carbamylation of deprotonated amines (10). The pH-independent second order rate constant of carbamyla- tion is also markedly dependent on the ionic strength pointing out the great electrostatic effect exerted by other protein residues in lowering (by several pH units) the pK of the E- amino group at the active site. It is most interesting that the ionic strength effects observed for the carbamylation of Lys- 258 are very similar to those detected for the pK dependence of the group influencing the chemical shift of the active site- bound phosphopyridoxyl trifluoroethylamine.

Trifluoroacetate Perfluorosuccinate

13 t 3 20 rt 5 1.0 k 0.2 2.0 k 0.3

The pH dependence of the lgF chemical shift of free phospho- pyridoxyl trifluoroethylamine has been found to be negligible in the pH range from 6.2 to 10 (10). On the other hand, the compound when bound to apoenzyme showed a downfield chemical shift as the pH increased (10). The interpretation that this is due to the direct influence of a group on the protein is invalid if instead the protein groups affect the pK of the secondary amine near the trifluoromethyl group. The pK of the amine as independently determined is 5.9 and this is detected as a downfield chemical shift change with decreasing pH. The direction of this pH-dependent chemical shift lead us to believe that the observed pa-dependent chemical shift changes of the fluorine at the active state are not due to the ionization of the secondary amine. These changes instead most likely reflect the ionization of a group which when carbamylated ceases to influence the electric field of fluorine.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

Carbamylation and lgF NMR of Active Site pK Determinations 2097

r

0

FIG. 5. Proposed relationship of active site-bound phosphopyri- doxyl trifluoroethylamine and the carbamylated e-amino group Lys- 258. The scheme also shows the proposed (12) anion binding site histidine at the active center.

Otherwise, regardless of the degree of carbamylation, the effect of the protein in the secondary amine should relate to the resonance on its nearby fluorine.

The observed T, values of protein-bound ‘“F are lower than would be expected from isotopic motion considerations only and any amount of internal rotation, which should be expected from a CF, group, would further increase the T, value. Thus, the low T, values most likely represent the intermolecular contribution to the lSF relaxation from nearby protons in the three-dimensional structure of the protein.

The intermolecular contribution of the lSF relaxation prob- ably involves more than 1 proton in the protein which, if it follows similar behavior to that in the fluorotyrosine of alka- line phosphatase (17), should be at a mean distance of 2 to 3 A from the fluorine nuclei. Thus, it is somewhat surprising that succinate, which binds to groups in the active center region, does not affect this interaction. One protein locus which has been ascribed to succinate binding is a histidyl residue. Our findings, therefore, could be interpreted as for the histidyl residue being further away than 3 A from the CF, moiety. This interpretation would certainly be consistent with the fact that all the ionization effects detected by the 13F probe can be accounted for by the ionization of a single residue which is not a histidyl residue (9).

Our assumptions regarding the interpretation of the T, data are solely based on the treatment of an unlike two-spin system, ‘H-‘“F. Since our probe is R-CH,-CFZ1, the possibility of deviation from this simpler treatment exists as the relaxa- tion rates of ‘“F nuclei may include terms for the transition rates of interacting multinuclear-like spins. However, the calculated molecular correlation time 7, using the TJT, data (80 ns) is similar to that value obtained by Churchich (19) for the transaminase using fluorescence depolarization tech- niques, and in the range expected for a protein of this size (20). From the expected values of 7, of 50 to 80 ns for this enzyme, we should have detected a ratio for T,/rrT, between 5 and 8. Our measured value of 6.4 for this ratio most encour- agingly favors our simple assumptions in the treatment of the relaxation data.

The nature of the protein group influencing the chemical

shift of the bound fluorinated pyridoxal analogue was tentativ- ely assigned by us to an amine group (9). The possibility that this group is the NH,-terminal is eliminated by the observa- tion of identical response of the resonance signal to pH changes in native enzyme or after extensive carbamylation of the exposed amines and of the NH,-terminal. By contrast, the additional carbamylation of Lys-258 results in a disappearance of the protein effect on the fluorine probe.

It must be concluded that the protein group with a pK of 8.3 influencing the Ic)F resonance of the active center-bound phos- phopyridoxyl trifluoroethylamine is most likely Lys-258. A model consistent with the appearance of the active site of aspartate transaminase is represented in Fig. 5. The ioniza- tion of Lys-258 would influence the resonance of the fluorines. This effect would be abolished when, as depicted, the lysyl residue is carbamylated. The active site histidyl residue, which binds inhibitory anions (211, appears to be too distant to influence the ‘!‘F probe as analysis of the pH titration of the chemical shift changes correspond to that of a single ionizing group (9). The coenzyme is also shown bound to hypothetical pockets in the protein; a cationic pocket for the phosphate group (16) and an anionic one for pyridine ions (2, 22). The native or physical properties (or both) of these pockets remains unknown.

1.

2.

3.

8.

9.

10.

11.

12.

13.

14.

15.

16. 17. 18 19 20

REFERENCES

Hughes, R. C., Jenkins, W. T., and Fisher, E. L. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 1815-1818

Braunstein, A. E. (1973) in The Enzymes (Bayer, P. D., ed) 3rd Ed, Vol. 9, pp. 379-481, Academic Press, New York

Polyanovsky, 0. L., and Torchinsky, Yu. M. (1963) in Chemical and Biological Aspects of Pyridoxal Catalysis (Snell, E. E., Fasella, P., Braunstein, A., and Rossi Fanelli, A., eds) pp. 157, Pergamon Press, New York

Stankewicz, M. J., Cheng, S., and Martinez-Carrion, M. (1971) Biochemistry 10, 2877-2884

Ivanov, V. I., Breusov, Yu. N., Karpeisky, M. Ya., and Poly- anovsky, 0. L. (1967) Mol. Biol. (Most.) 1, 588-595

Martinez-Carrion, M., Tiemeier, D. C., and Peterson, D. L. (1970) Biochemistry 9, 2574-2582

Yang, I.-Y., Harris, C. M., Metzler, D. E., Korytnyk, W., Lachmann, B., and Potti, P. P. G. (1975) J. Biol. Chem. 250, 2947-2955

Martinez-Carrion, M., Turano, C., Riva, F., and Fasella, P. (1967) J. Biol. Chem. 242, 1426-1430

Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio, A. M. (1976) J. Biol. Chem. 251, 1853-1858

Slebe, J. C., and Martinez-Carrion, M. 11976) J. Biol. Chem. 251, 5663-5669

Feliss, N., and Martinez-Carrion, M. (1970) Biochem. Biophys. Res. Commun. 40, 932-940

Martinez-Carrion, M., Cheng, S., and Relimpio, A. M. (1973) J. Biol. Chem. 248, 2153-2160

Veronese, F. M., Piszkiewicz, D., and Smith, E. L. (1972) J. Biol. Chem. 247, 754-759

Garner, M. H., Bogardt, R. A., Jr., and Gurd, F. R. N. (1975)J. Biol. Chem. 250, 4398-4404

Relimpio, A., Slebe, J. C., and Martinez-Carrion, M. (1975) Biochem. Biophys. Res. Commun. 63, 625-634

Martinez-Carrion, M. (1975) Eur. J. Biochem. 54, 39-43 Hull, W. E., and Sykes, B. D. (1974) Biochemistry 13, 3431-3437 Hull, W. E., and Sykes, B. D. (1978)J. Mol. Biol. 98, 121-153 Churchich, J. E. (1967) Biochim. Biophys. Acta 147, 511-517 Yguerrabide, J., Eptein, H. F., and Stryer, L. (1970) J. Mol.

Biol. 51, 573-590 21. Martinez-Carrion, M., Kuczenski, R., Tiemeier, D. C., and

Peterson, D. L. (1970) J. Biol. Chem. 245, 799-805 22. Fasella, P. (1968) in Pyridozal Catalysis: Enzymes and Model

Systems. (Snell, E. E., Braunstein, A. E., Severin, E. S., and Torchinsky, Yu. M., eds) pp. 1-31, John Wiley and Sons, New York

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

J C Slebe and M Martinez-Carrionto the active site lysyl residue in aspartate transaminase.

Selective chemical modification and 19F NMR in the assignment of a pK value

1978, 253:2093-2097.J. Biol. Chem. 

  http://www.jbc.org/content/253/7/2093.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/253/7/2093.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from