THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. E, Issue of March 15, pp. 546”5470,1993 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U.S.A. Mutational Analysis of Active Site Residues of Human Adenosine Deaminase* (Received for publication, September 8, 1992) Dipa Bhaumik, Jeffrey Medin$, Karen Gathy, and Mary Sue Coleman8 From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 Adenosine deaminase was overexpressed in a bacu- combined immunodeficiency (Giblett et al., 1972). These pa- lovirus system. The pure recombinant and native en- tients have no obvious gastrointestinal tract abnormalities, zymes were identical in size,Zn2+ content, and activity. but they do exhibit a dramaticlymphopenia that seems to be Five amino acids, in proximity to the active site, were a direct consequence of the absence of adenosine deaminase replaced by mutagenesis. The altered enzymes were (Coleman et al., 1978; Donofrio et al., 1978). purified to homogeneity and compared to wild-type Potent inhibitors of adenosine deaminase are lympholytic adenosine deaminase with respect to zinc content, en- in humans, and this property hasbeen exploited in the treat- zymatic activity, and kinetic parameters. All but one ment of certain leukemias, the hallmark of which is accumu- of the alterations produced significant activitypertur- lation of differentiation-arrested lymphocytes (Coleman, bations. Replacement of Cysz02produced a protein that 1983). Ground and transition stateanalog inhibitors have also retained at least 30-40% of wild-type activity.In con- proven useful in studies of the reaction mechanism of aden- trast, replacements of His17, His214,Hiszs8, and Glu217 resulted in dramatic losses of enzyme activity. None of osine deaminase. With a rate enhancementof about lo‘’, this these mutants exhibited large variations in K,. The enzyme is among the most efficient that have been described proteins produced from alterations of amino acids im- (Frick et al., 1987). A hydrate tetrahedral intermediate has plicated in metal coordination were slightly activated been postulated from a large number of chemical studies by inclusion ofZnz+ throughout purification. These (Evans and Wolfenden, 1973; Wolfenden et al., 1969; Kurz experiments confirm that in the active enzyme Zn2+ and Frieden, 1983). The most convincing evidence for this plays a critical role in catalysis, that a histidine or intermediate reaction product is from 13C NMR studies of glutamate residue plays a mechanistic role in the hy- adenosine deaminase bound to purine riboside (1,6-dihydro- drolytic deamination step, and that cysteine is not in- purine riboside), in which a change of hybridization from sp2 volved in the catalytic mechanism of adenosine deam- to sp3 is detected (Kurz and Frieden, 1987). Subsequent UV inase. These data support the roles for these amino acid and NMR studies confirmed that this inhibitor is bound as residues suggested from the x-ray structure of murine an oxygen adduct, presumably hydrated at the 1,6 position adenosine deaminase (Wilson, D. K., Rudolf, F. B., and (Jones et al., 1989). This covalent hydrate with C6 in the Quicho, F. A. (1991)Science 252, 1278-1284). adenosine deaminase-purine riboside complex has been con- firmed recently by the determination of its structure by x-ray crystallography (Wilson et al., 1991). Unexpectedly, the crys- talstructure also revealed that adenosine deaminase is a Adenosine deaminase (EC 3.5.4.4), an important enzyme of metalloenzyme that complexes 1 mol of Zn2+ per molof the purine salvage pathway, catalyzes the irreversible hydro- protein. lytic deamination of adenosine or deoxyadenosine to inosine Solution of the crystal structure of a mammalian adenosine or deoxyinosine. Adenosine deaminase is expressed at very deaminase provided knowledge of the amino acids in the high levels along the entire murine gastrointestinal tract, in active site. However, at pH4.2, where crystals were generated thymic T cells and in decidual cells of the developing mater- for x-ray analysis, adenosine deaminase is almost completely nal-fetal interface (Lee, 1973; Knudsen et al., 1988 and 1989; inactive, and at this pH thesubstrate analogue, purine ribo- Witte et al., 1991). In humans,the upper gastrointestinal tract side is only weakly bound (Wolfenden and Kati, 1991). Con- is devoid of this enzyme activity, but high levels are expressed struction of mutations in active site residues coupled with in the lower part of the tract. determination of functional consequences of each mutation The wide spectrum of adenosine deaminase activity in under conditions of optimal enzyme activity, will permit de- mammalian tissues portended an important role for purine tailed characterization of the reaction pathway and descrip- metabolism in nutrition and reproduction. However, the en- tion of enzyme intermediates. tirepurine salvage pathway, and adenosine deaminase in In this study, guided in selection of targets by the crystal particular, became the focus of intenseinterest with the structure, we have altered key amino acid residues within the observation that hereditary deficiency of the enzyme in hu- active site of human adenosine deaminase, an enzyme that is man infants is invariably associated with a form of severe highly homologous to its murine counterpart. The recombi- nant enzymes were expressed in a baculovirus system and * This work was supported in part by United States Public Health purified to homogeneity on a monoclonal antibody affinity Service Grant CA26391 (to M. S. C.). The costs of publication of this column. Kinetic characteristics,stabilities, and metal binding article were defrayed in part by the payment of page charges. This capacities of the altered enzymes were assessed and correlated article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. with mechanistic models. $ Recipient of an Army Predoctoral Fellowship in Biotechnology. Present address: Laboratory of Molecular Growth Regulation, EXPERIMENTALPROCEDURES NICHD, National Institutes of Health, Bethesda, MD 20892. Materials-Oligodeoxynucleotide primers used in constructing mu- § To whom correspondence should be addressed. tations and sequencing were synthesized at the University of Ken- 5464 This is an Open Access article under the CC BY license. MutationalAnalysis of AdenosineDeaminase 5465 tucky Macromolecular Structure Facility on an Applied Biosystems stage. The infection was allowed to continue for 4 days after which 380B DNA synthesizer. Restriction endonucleases were from United the larvae were collected and frozen immediately at -70 "C. The States Biochemicals and New England Biolabs. Sequenase DNA mutants H17A and H214L were overexpressed in Sf-9 and High-5 sequencing kits were obtained from United States Biochemicals. [a- cells. For cellular infection, 2.5 X 107cellsin T-175 flasks were infected 36S]dATPand [14C]adenosinewere Du Pont-New England Nuclear at a multiplicity of infection of 10 with the appropriate virus stock. products. The polyclonal antibody used in these experiments was Cells were harvested 65-h postinfection, washed twice with cold raised inrabbits in our laboratory against homogeneous human phosphate-buffered saline, and frozen at -70 "C. adenosine deaminase. The anti-adenosine deaminase monoclonal an- Purification of Recombinant Proteins-Wild-type adenosine de- tibody (NlD1) used in the study was also generated in our laboratory aminase was purified from frozen larvae by adenosine-affinity chro- and propagated in ascitesfluid (Philips etal., 1987).All other reagents matography (Medin et al., 1990). Briefly, frozen larvae (28 g) contain- were of the highest commercial grade available. ing the recombinant protein were homogenized in a buffer (10 mM Bacterial Strains and Vectors-The Escherichia coli strains used sodium acetate, pH 6.4, 2 mM EGTA, 5 mM benzamidine, 10 mM 6- for plasmid propagation were CJ236 and DH5a (Bethesda Research aminocaproic acid, 5 mM phenylmethylsulfonyl fluoride), and centri- Laboratories). The plasmid vector M13 mp18 was used for site- fuged at 30,000 X g for 30 min. Protamine sulfate was added to the directed mutagenesis of the adenosine deaminase cDNA. The bacu- crude extract and allowed to precipitate. The clarified supernatant lovirus transfer vector pAcC4, a generous gift from Cetus, was used obtained after centrifugation was loaded onto a DEAE-Sephadex in homologous recombination experiments to construct specific bac- column (25 X 5 cm). The protein was eluted with the same buffer ulovirus variants. containing 0.5 M sodium chloride and concentrated by ammonium Viruses, Cells, and Larvae-Autographa californica nuclear poly- sulfate precipitation. The precipitate, resuspended ina minimum hedrosis virus (ACMNPV strain Li, Invitrogen Corp.) and Spodoptera volume (-8 ml) of phosphate-buffered saline, pH 7.4, was applied to frugiperda (Sf-9) insect cells (Invitrogen) used were propagated in the an adenosine-Sepharose column (110 X 1.5 cm) (Schrader and Stacy, laboratory and used in the protein expression experiments. The 1977), and the fractions containing the major adenosine deaminase second insect cell line, High-5 (Invitrogen), was derived from eggs of activity were pooled and concentrated by ultrafiltration. the cabbage looper and was an alternate host for recombinant bacu- Mutant proteins were isolated by using a monoclonal antibody lovirus propagation.