©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Analysis of a Recombinant UDP-N-acetyl-D-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase (*)

Stephanie Wragg , Fred K. Hagen , Lawrence A. Tabak (§)

From the (1)Departments of Dental Research and Biochemistry, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A mammalian expression vector was designed to express a secreted soluble form of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) with a metal binding site (HHWHHH) at the NH terminus. The recombinant enzyme was purified to homogeneity from COS-7 cell media by sequential chromatography on columns of NiCl-chelating Sepharose, Affi-Gel blue, and Sephacryl S-100. Kinetic parameters of recombinant and native polypeptide GalNAc transferase were comparable for the donor UDP-GalNAc and for the peptide acceptors AcTPPP, EPO-T (PPDAATAAPLR), and HVF (PHMAQVTVGPGL). Initial velocity and product inhibition studies were carried out with purified recombinant polypeptide GalNAc transferase and the substrates UDP-GalNAc and peptide EPO-T. Initial velocity data was consistent with a sequential type mechanism in which binding of both substrates precedes product release. Product inhibition analysis using UDP showed competitive inhibition against UDP-GalNAc and a noncompetitive inhibition against peptide EPO-T. The dead end peptide analogue EPO-G (PPDAAGAAPLR) was a noncompetitive inhibitor of UDP-GalNAc and a competitive inhibitor of peptide EPO-T. Collectively, the results suggest that the most probable kinetic mechanism for the enzyme is one in which both substrates must bind in a random order prior to catalysis. Interestingly, the K for EPO-T is similar to the K for EPO-G, suggesting that peptide interaction with the polypeptide GalNAc transferase does not require a hydroxyamino acid.


INTRODUCTION

The initial step in mucin type O-linked glycosylation is catalyzed by the enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41) (polypeptide GalNAc transferase)()and results in the covalent attachment of GalNAc to the hydroxyamino acids threonine and serine in an -anomeric linkage. The two-substrate/two-product reaction utilizes the nucleotide sugar UDP-GalNAc and a polypeptide acceptor and produces free UDP and glycosylated protein in a yet undefined mechanism(1) . The polypeptide GalNAc transferase is the first enzyme in a series of glycosyltransferases leading to the stepwise assembly of protein-linked complex carbohydrate structures (2). As such, it represents a potentially important early control point in O-glycosylation, and it is therefore important to define its mechanism.

Previous characterization of the enzyme has demonstrated that the monomeric soluble enzyme requires the divalent cation manganese for its activity(3, 4, 5) . The donor specificity is restricted to UDP-GalNAc (3, 5); in contrast, substrate specificity studies indicate that the enzyme binding site is large (8 or 9 residues) and can accommodate a wide range of amino acids that flank the glycosylation site(6, 7) . However, the kinetics of the enzyme in terms of the mechanism and the order in which the two substrates bind have not yet been defined.

Available evidence suggests that more than one polypeptide GalNAc transferase may exist and that these may exhibit overlapping specificities(5, 8, 9, 10) . Therefore, in the present study, we have purified to apparent homogeneity a recombinant form of this enzyme. We have measured kinetic parameters of this enzyme; these are consistent with the polypeptide GalNAc transferase binding to its substrates in a random sequential order prior to catalysis. We further demonstrate that the presence of a hydroxyamino acid is not essential for the interaction of the polypeptide GalNAc transferase with peptide substrates.


EXPERIMENTAL PROCEDURES

Plasmid Construction

An insulin secretion signal sequence (gift of Dr. K. Drickamer) and the coding region of the bovine placental polypeptide GalNAc transferase were introduced into the mammalian expression vector pSVL (Pharmacia Biotech Inc.) as described in Hagen et al.(9) . Synthetic oligonucleotides were used to engineer a metal binding site with the amino acid sequence HHWHHH (modified from Ref. 11) immediately downstream from the insulin processing site (Fig. 1A). The secreted recombinant enzyme encoded by plasmid pSVL-inGNT1 is thus designed with an in vivo cleavable processing site that produces the NH-terminal amino acid sequence FVHMHHWHHH preceding the native soluble form of the polypeptide GalNAc transferase.


Figure 1: Sequence of the NH-terminal region of the recombinant polypeptide GalNAc transferase. A, nucleotide and amino acid sequence of the first 156 nucleotides of the recombinant polypeptide GalNAc transferase. The first 78 nucleotides encode the insulin secretion signal from pGIR-199 (19). Changes introduced in the bovine cDNA during cloning are underlined. The remainder of the cDNA (not shown) is identical to that reported in Ref. 9. The metal binding site is boxed. The arrow points to the insulin signal peptide processing site. B, NH-terminal amino acid sequence of the secreted recombinant polypeptide GalNAc transferase. The first 14 amino acids were determined experimentally, while the remainder were inferred from the conceptually translated sequence. C, NH-terminal sequence of the secreted form of the naturally secreted bovine colostrum GalNAc transferase enzyme (9).



Expression of the Recombinant Polypeptide GalNAc Transferase

Plasmid pSVL-inGNT1 was used to transfect COS-7 cells. COS-7 cells were grown to 80-90% confluence (approximately 1 10 cells) at 37 °C in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum, 6% CO in 10 100-mm culture dishes. Cells were transfected with 8 µg DNA using a liposome-mediated transfection protocol (8 µl of LipofectAMINE Reagent, Life Technologies, Inc.). At 72 h post-transfection, the media were harvested and replaced with conditioned Dulbecco's modified Eagle's medium. At 96 h post-transfection, the media were harvested.

Purification of Recombinant Polypeptide GalNAc Transferase from COS-7 Cell Supernatants

Sterile buffers used in the enzyme purification were SBB (30 mM MOPS, 10 mM MgCl, 100 mM NaCl, 20 mM imidazole, pH 6.5), wash buffer A (50 mM MES, 100 mM NaCl, 10 mM MgCl, pH 5.7), wash buffer B (100 mM MES, 100 mM NaCl, 10 mM MgCl, pH 4.7), binding buffer (30 mM MOPS, 10 mM MgCl, 100 mM NaCl, pH 7.1), buffer D (25 mM MES, 5 mM MnCl, 0.001% Triton X-100 (hydrogenated form), pH 6.5), and buffer E, 25 mM MES, 80 mM NaCl, 10% glycerol, 0.05% Triton X-100 (hydrogenated form), pH 6.5.

The harvested media were clarified of cellular debris by low speed centrifugation and diluted to SBB conditions (150 ml). The pool was applied to columns of NiCl-chelating Sepharose (Pharmacia) equilibrated with SBB and fractionated as described in the legend to Fig. 3.


Figure 3: Purification of the recombinant polypeptide GalNAc transferase. All fractions were monitored for absorbance at 280 nm (hatchedline) and assayed for transferase activity (closedcircles). A, harvested media were loaded on a column of NiCl-chelating Sepharose (1.5 20 cm) equilibrated with SBB. The column was developed sequentially with a 32-ml linear gradient of wash buffer A to B (arrow 1), 34 ml of wash buffer B (arrow 2), and 70 ml of binding buffer, 50 mM imidazole (arrow 3). Bound transferase was eluted with 35 ml of binding buffer, 250 mM imidazole (arrow 4). Enzyme-containing fractions were pooled and diluted 1:3 in buffer D. B, the enzyme-containing material was loaded onto a column of Affi-Gel blue (0.7 10 cm) equilibrated with buffer D. The column was developed sequentially with 10 ml of buffer D, 100 mM NaCl (arrow 1) and 15 ml of buffer D, 100 mM NaCl, 20% glycerol (arrow 2). Bound transferase was eluted from the column with a 16-ml linear salt gradient of 0.1-1 M NaCl in buffer D and 20% glycerol (arrow 3). The enzyme-containing fractions were pooled and dialyzed against buffer E. C, the dialyzed material was concentrated 20-fold using a Centricon-30 (Amicon) and loaded onto a Sephacryl S-100 gel filtration column (1 120 cm) equilibrated in buffer E. The active fractions were pooled, concentrated 41-fold using a Centricon-30, supplemented with sodium azide to 0.02%, and stored at -70 °C.



Protein Determination and Amino Acid Analysis

Prior to analysis, aliquots from each purification step were buffer-exchanged for water using Micron 10 ultrafiltration devices (Amicon). The proteins were then hydrolyzed under standard conditions (vapor phase HCl, 106 °C, 20 h), and the hydrolyzed material was analyzed on a Hewlett-Packard amino acid analyzer with Amino Quant II software.

SDS-PAGE of Protein

Aliquots of the pools from the recombinant transferase purification were subjected to 10%/3% Tricine SDS-polyacrylamide gel electrophoresis(12) . To detect proteins, samples were dried down and resuspended in 100 mM Hepes, 6 M urea, 20 mM NaCNBH, and a 10-fold excess of [C]formaldehyde to lysine in protein(13) . After incubation at 37 °C for 24 h, the samples were ethanol-precipitated (80%) and resuspended in Tricine SDS-PAGE gel loading buffer (4% SDS, 12% glycerol (w/v), 50 mM Tris, pH 6.8, 2% mercaptoethanol (v/v), 0.01% Serva Blue G). Electrophoresis of 10% gels was performed at 15-20 mA and was followed by soaking of the gel in EnHance (DuPont NEN). The gel was dried down, and radiolabeled protein was visualized by autoradiography on Kodak XAR film.

Amino Acid Sequence Data

To obtain NH-terminal amino acid sequence data, 8 pmol of purified recombinant transferase from step 3 was exchanged into water using a Centricon-10 (Amicon) ultrafiltration device. The sample was applied to a Polybrene-coated trifluoroacetic acid-activated precycled glass fiber filter and sequenced on an Applied Biosystems 473A protein sequencer.

Determination of Polypeptide GalNAc Transferase Activity and Kinetic Parameters

The recombinant polypeptide GalNAc transferase was assayed for synthesis of GalNAc-substituted peptide in 25-50-µl reactions containing 40 mM sodium cacodylate, 0.1% Triton X-100, 4 mM -mercaptoethanol, 10-15 mM MnCl, pH 6.5. The acceptor peptides EPO-T (PPDAATAAPLR), HVF (PHMAQVTVGPGL), and AcTPPP have been described previously (8) and used in a concentration range of 0.02-5 mM. The UDP-[C]GalNAc (DuPont NEN) and UDP-GalNAc (Sigma) concentration range was 1-1600 µM. The UDP (Sigma) stock solution concentration was determined spectrophotometrically (extinction coefficient (260 nm) = 10 optical density units/mM). The purified recombinant transferase was diluted 1:20 in 50 mM cacodylate, 100 mM NaCl, 50% glycerol, pH 6.5 added to the reactions. The reactions were incubated at 37 °C for a period of time such that less than 10% of the substrate UDP-[C]GalNAc was consumed. At this point, the incubation was terminated by addition of 10 µl of 150 mM EDTA and passed through anion exchange spin columns (Dowex AG 1-X8, Bio-Rad), and the counts were determined by liquid scintillation spectroscopy. The kinetic experiments were performed at least twice.

Data were plotted graphically to show the initial velocity and fitted to Equation 1, corresponding to the initial velocity of a two-substrate reaction(14) .

On-line formulae not verified for accuracy

where V is the maximum velocity, K is the Michaelis constant for the varied substrate, and A and B are the concentrations of A and B.

Data were plotted graphically to show inhibition patterns and fitted to Equations 2-4 corresponding to competitive, noncompetitive, and uncompetitive inhibition, respectively(14) .

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

where I is the concentration of inhibitor I, K is the Michaelis constant, and K and K are the slope and intercept constants, respectively. Starting estimates of the parameters were obtained from linear regression, and fits were made using the NLIN procedure of SAS with the Gauss-Newton method(15) .


RESULTS

Expression of the Recombinant Polypeptide GalNAc Transferase

The plasmid construct pSVL-inGNT1 contains an insulin secretion signal preceding a metal binding site and polypeptide GalNAc transferase coding sequence as defined in Fig. 1A. In vivo processing of the signal peptide in COS-7 cells directs secretion of the soluble form of the recombinant transferase with an NH-terminal metal anchor (Fig. 1B) into the culture medium. COS-7 cells transiently transfected with pSVL-inGNT1 expressed and secreted sufficient GalNAc transferase, such that 1 µl of culture medium incorporated 2,000-10,000 cpm/h of UDP-[C]GalNAc into peptide substrates under standard assay conditions. Splitting cells 18 h post transfection resulted in a reduction in the concentration of expressed enzyme activity relative to unsplit cells. Growth at 100% confluence for 5 days was not detrimental to this transient expression system (Fig. 2). In an experiment to determine the duration of gene expression, maximal expression of the recombinant gene was achieved at 48 h post-transfection, whereas de novo recombinant enzyme synthesis was minimal by 96 h (data not shown), indicating that the plateau for enzyme activity observed in Fig. 2was due to a decrease in gene expression and not to instability or degradation of the recombinant enzyme. Indeed, the soluble recombinant enzyme was stable at 37 °C for at least 3 days, since a 37 °C incubation of the clarified culture medium did not result in loss of recombinant enzyme activity (data not shown).


Figure 2: Transient expression of a soluble recombinant polypeptide GalNAc transferase from COS-7 cells. Enzyme activity duplicate measurements were made at 18, 40, 63, 117, and 168 h post transfection. Opensymbols indicate cells grown to 100% confluence and not split. Closedsymbols indicate cells split 1:6 18 h post-transfection. Media were clarified, and 1 µl was assayed for activity under standard conditions using acceptor peptide AcTPPP.



Recombinant Polypeptide GalNAc Transferase Purification

A summary of the purification of this enzyme from approximately 10 COS-7 cells is given in . Metal chelate chromatography (step 1) was used to selectively remove the recombinant form of the transferase from the media supernatants by virtue of the affinity tag (HHWHHH) at the NH terminus of the enzyme (Fig. 3A). Chromatography on Affi-Gel blue (step 2) eliminated high molecular weight contaminants (Fig. 4, lane3) and increased the specific activity of the material 7-fold (Fig. 3B). Gel filtration on Sephacryl S-100 (step 3) (Fig. 3C) yielded a recombinant transferase fraction that migrated as a single band on Tricine SDS-PAGE (Fig. 4, lane4). The apparent molecular weight of the recombinant GalNAc transferase was 68,000 Da, a value consistent with that of the native bovine colostrum GalNAc transferase(9) . The NH-terminal sequence of the recombinant polypeptide GalNAc transferase was determined by NH-terminal protein sequencing (Fig. 1B). The presence of the processed insulin secretion signal and the metal binding sequence indicate that the protein was intact.


Figure 4: 10%/3% Tricine SDS-PAGE analysis of reductively C-methylated aliquots of pooled fractions. Lane 1, COS-7 cell supernatant; lane 2, pooled material from metal chelate chromatography; lane 3, pooled material from Affi-Gel blue; lane 4, pooled material from gel filtration; lane 5, bovine serum albumin control as size marker; lane 6, C-labeled molecular weight markers.



Kinetic Properties of the Recombinant Polypeptide GalNAc Transferase

To determine whether the addition of a metal binding domain and secretion by COS-7 cells into tissue culture medium had any effect on the properties of the enzyme, kinetic parameters K and V of the purified recombinant GalNAc transferase were compared with those of the purified native enzyme (). The K and V for UDP-GalNAc were obtained with peptide AcTPPP at saturating conditions with both enzymes; the recombinant GalNAc transferase displayed an acceptor specificity similar to that of the native GalNAc transferase with regard to the Kvalues for the nucleotide sugar and for peptides AcTPPP and HVF. Similarly, the V values for both the nucleotide sugar and peptide AcTPPP were the same. These results indicated that the kinetic properties of the recombinant polypeptide GalNAc transferase were not affected by the presence of the metal binding domain at its NH terminus.

Initial Velocity Pattern

Two-substrate enzyme reactions can proceed by one of three mechanisms: (a) ping-pong, (b) compulsory ordered sequential, or (c) random ordered sequential(17) . To identify the mechanism preferentially used by the polypeptide GalNAc transferase, experiments were performed in which the concentrations of peptide EPO-T and UDP-GalNAc were varied. Six concentrations of UDP-GalNAc ranging from 1 to 80 µM and six concentrations of peptide EPO-T ranging from 0.07 to 2 mM (0.2-8 K for UDP-GalNAc and 0.2-5 K for EPO-T) were used. Concentrations of peptide beyond 2 mM were avoided because problems of peptide solubility arise and because the velocity of the reaction begins to decrease at values slightly greater than 2 mM. The data were graphed in a double-reciprocal plot (Fig. 5) and produced a family of intersecting lines typical of a sequential mechanism. A plot of 1/vversus 1/[EPO-T] also produced a family of intersecting lines (data not shown). Data obtained were fit to the initial velocity equation (Equation 1), which can be modified to specifically describe each of the two-substrate enzyme reaction mechanisms outlined above. The ping-pong model is a special case of Equation 1 in which K = 0(14) . When the K is 0, the slope expression of the double-reciprocal form of Equation 1 becomes insensitive to changes in the concentration of the second substrate in the reaction. On a double-reciprocal plot, this produces the typical parallel line pattern of the ping-pong mechanism. The estimate of K (I) was found to be more than 6.7 standard errors away from 0 (95% confidence interval (0.0111, 0.0207)) and is thus inconsistent with the ping-pong mechanism. The equilibrium-ordered mechanism is also a special case of Equation 1 in which K = 0(14) . However, the estimate of K (I) was found to be more than 6.1 standard errors away from 0 (95% confidence interval (0.0067, 0.0133)), and this is inconsistent with the equilibrium-ordered sequential model.


Figure 5: Double-reciprocal plot of initial velocity versus UDP-GalNAc concentration at fixed variable EPO-T concentrations. Peptide EPO-T concentration range was 0.2-5 K (0.07-2 mM), and UDP-GalNAc concentration range was 0.2-8 K (1-80 µM). The assays were performed as described under ``Experimental Procedures.''



Product/Dead End Inhibition

To further distinguish between an ordered sequential and random mechanism, we first examined the inhibitory effect of UDP. UDP is a reaction product known to inhibit the reaction(3) . Fig. 6A shows that UDP was a competitive inhibitor of UDP-GalNAc. The slope effect, combined with the absence of an intercept effect, typical of competitive inhibition, was further emphasized by the replot (Fig. 6A, inset). Fig. 6B shows the effect of UDP with peptide EPO-T as the varied substrate. The replot (Fig. 6B, inset) shows both a slope and intercept effect consistent with UDP being a noncompetitive inhibitor of the peptide.


Figure 6: Product inhibition by UDP with variable UDP-GalNAc concentrations (A) and with variable EPO-T concentrations (B). Substrates and the inhibitor UDP were added simultaneously to the enzyme, and assays were performed as described under ``Experimental Procedures.'' In A, the peptide EPO-T concentration was 0.4 mM. UDP concentrations were 0 (), 0.056 (), 0.142 (), 0.28 (), 0.7 (⊞), and 2 mM (). In B, the UDP-GalNAc concentration was 20 µM. UDP concentrations were 0 (), 0.037 (), 0.23 (), 0.46 (), 1.15 (⊞), and 2 mM (). Insets show secondary replots of slopes and intercepts versus UDP concentration.



To unambiguously distinguish between a random ordered and ordered sequential mechanism, a dead end peptide analogue in which the threonine residue of EPO-T was replaced with a glycine residue (EPO-G: PPDAAGAAPLR) was used in inhibitor studies. If UDP-GalNAc participates in an ordered sequential mechanism, a pattern of uncompetitive inhibition would be seen when the concentrations of UDP-GalNAc and EPO-T were varied and held constant, respectively. This would graphically translate as a family of parallel lines on a double-reciprocal plot. However, Fig. 7A shows that a series of parallel lines is not generated. The replot (Fig. 7A, inset) indicates that there was a slope effect as well as a slight intercept effect providing evidence against an ordered sequential mechanism. The statistical fit of the data was consistent with EPO-G being a noncompetitive inhibitor of UDP-GalNAc (I). At a fixed subsaturating concentration of UDP-GalNAc, only changes in slope consistent with competitive inhibition were observed with varying concentrations of EPO-G (Fig. 7B and inset). The absence of uncompetitive inhibition between the dead end analogue EPO-G and either of the substrates ruled out an ordered mechanism in which there is compulsory binding of one substrate before the other to form a ternary complex. Instead, the patterns of inhibition of the dead inhibitor EPO-G were consistent with a random sequential mechanism.


Figure 7: Dead end substrate inhibition by peptide EPO-G with variable UDP-GalNAc concentrations (A) and variable peptide EPO-T concentrations (B). Substrates and the peptide inhibitor EPO-G were added simultaneously to the enzyme, and assays were performed as described under ``Experimental Procedures.'' In A, the peptide EPO-T concentration was 0.4 mM. EPO-G concentrations were 0 (), 0.18 (), 0.4 (), 1 (), 2 (⊞), and 4 mM (). In B, the UDP-GalNAc concentration was 20 µM. EPO-G concentrations were 0 (), 0.15 (), 0.4 (), 1 (), 1.5 (⊞), 2.5 (), and 4 mM (). Insets show secondary replots of slopes and intercepts versus peptide EPO-G concentration.




DISCUSSION

We have transiently expressed a secreted recombinant form of a polypeptide GalNAc transferase indistinguishable from the native enzyme with respect to kinetic parameters (K, V). Since several lines of evidence have suggested that the polypeptide GalNAc transferase may be expressed in multiple forms with overlapping specificities, the characterization of a homogeneous form of the enzyme represents an initial step in determining if alternate forms of this enzyme exist. An insulin secretion signal was used to target the recombinant enzyme for secretion(19) , thereby allowing us to distinguish the cloned activity from the endogenous activity of COS-7 cells. The metal binding affinity tag engineered in the amino terminus of the recombinant enzyme provided a facile method to remove over 98% of contaminant proteins from cell culture media by metal chelate chromatography. Approximately 5 µg of homogeneous recombinant polypeptide GalNAc transferase was prepared from the culture supernatants of 10 COS-7 cells.

Our data suggest that the polypeptide GalNAc transferase reaction proceeds via a random ordered sequential mechanism (Fig. S1). This mechanism is consistent with the observation that bovine colostrum polypeptide GalNAc transferase can be purified on a 5-Hg-UDP-GalNAc affinity matrix in the absence of protein substrate (9). Further, the estimated kinetic constants K and K for UDP-GalNAc (10 µM, 16 µM) and EPO-T (0.408 mM, 0.648 mM) were similar to one another (I); the similarity of the K and K of a substrate is an indication that their binding to the enzyme occurs independently of one another(17) .


Figure S1: Scheme 1.



To our knowledge, only one other nucleotide sugar:protein transferase has been described in terms of reaction mechanism. UDP-D-xylose:core protein -D-xylosyltransferase catalyzes the first step in chondroitin sulfate proteoglycan O-glycosylation. The reaction in which xylose, donated by the nucleotide sugar UDP-xylose, is added to serine has been determined to proceed via an ordered sequential mechanism(20) . It remains unclear why two enzymes that catalyze similar committed steps of O-glycosylation do so using different mechanisms.

In this study, the K of UDP was 380 µM, as determined by slope and intercept effects (I). This value is one order of magnitude greater than the K of a polypeptide GalNAc transferase purified from an ascites hepatoma and published previously (26 µm; Ref. 3). The discrepancy might be because the two enzymes represent different proteins. The effect of UDP on the activity of the polypeptide GalNAc transferase described here was not as potent as for the polypeptide GlcNAc transferase catalyzing the addition of GlcNAc residues to hydroxyamino acids; more than 50% of the activity of this cytosolic enzyme could be abolished by as little as 0.5 µM UDP(21) . Although both nucleotide sugar:protein transferases glycosylate polypeptides via the transfer of a saccharide donated by a UDP-based nucleotide-sugar, the data here suggested that UDP may not be an important means of regulation of polypeptide GalNAc transferase activity. However, similar to the lack of effect of free GlcNAc on the activity of the polypeptide GlcNAc transferase(21) , the presence of various concentrations of free GalNAc had no significant effect on the activity of the polypeptide GalNAc transferase (data not shown), suggesting a requirement by the enzyme for GalNAc to be attached to a uridine diphosphate.

The data from the competitive inhibition between EPO-T and EPO-G provided insight into the mechanism of site selection of the enzyme. The similarity of the K value for EPO-T (0.408 mM) and the K value for the dead end inhibitor EPO-G (0.770 mM) indicates that the enzyme binds to both peptides with similar affinity. Substrate selection may be a two-part process in which the enzyme can bind to a substrate that meets the flanking sequence requirements, followed by binding to the target itself, i.e. the hydroxyamino acid. We speculate that the competition observed in vitro between EPO-T and EPO-G can occur in vivo in that both hydroxyamino acid-bearing peptides and peptides devoid of hydroxyamino acids can bind to the enzyme but can also exchange with one another, thus creating a potential point of regulation. We are currently conducting studies, in vivo, to test this hypothesis.

  
Table: 0p4in Due to the small amount of material available for analysis, the total protein was estimated by averaging the results of amino acid analysis (12.6 µg), NH-terminal sequencing initial yield values (2.3 µg), and scanning densitometry (1.03 µg) of Fig. 4.

  
Table: Estimated kinetic constants for the recombinant polypeptide GalNAc transferase substrates

All assays were carried out as described under ``Experimental Procedures.''


  
Table: Estimated kinetic constants for the recombinant polypeptide GalNAc transferase



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DE-08108 (to L. A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Depts. of Dental Research and Biochemistry, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave., Box 611, Rochester, NY 14642. Tel.: 716-275-0770; Fax: 716-473-2679; E-mail: ltab@bphvax.biophysics.rochester.edu.

The abbreviations used are: polypeptide GalNAc transferase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Eric Phizicky for thoughtful comments about this work, Brian VanWuyckhuyse for the amino acid analysis and NH-terminal sequencing, Dr. Richard Raubertas for help with the statistical analysis, and Pat Noonan for preparing the manuscript.


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