GENOMICS

11,1014-1024

(1991)

Mammalian Hexokinase 1: Evolutionary Conservation and Structure to Function Analysis L. D. GRIFFIN,* B. D. GELB,*‘t

D. A. WHEELER,+

*Institute for Molecular Genetics, tDepartment Medicine, and §Department of Biochemical

D. DAVISON,~

Press, Inc.

INTRODUCTION Hexokinase (HK E.C.2.7.1.1) catalyzes the first step in glucose metabolism, utilizing ATP for the phosphorylation of glucose to glucose-6-phosphate. In mammals there are four HK isozymes, which vary in their tissue distribution and kinetic properties (Katzen and Schimke, 1965). Each of the types l-3 HK isozymes consists of a single polypeptide chain with a molecular weight of approximately 100 kDa and is inhibited by the product glucose g-phosphate (Katzen and Schimke, 1965). The type 4 HK, or glucokinase, is similar to yeast HK, as it is approximately 50 kD, and insensitive to inhibition by glucose-6-phosphate (Grossbard and Schimke, 1966). 1014 Inc. reserved.

E. R. B. MCCABE*‘t

18, 1991

Several groups (Ureta, 1982; Vowles and Easterby, 1979; Holroyde and Trayer, 1976) have speculated that the mammalian HKs evolved through the duplication and fusion of an ancestral hexokinase, which resembled the yeast HKs and mammalian glucokinase in size. After the duplication event, one of the halves evolved to have a regulatory function, while the other half retained the catalytic functions of the ancestral enzyme. The new HK gene then underwent further duplications to produce the three lOO-kDa HKs. It has also been suggested (Ureta, 1975) that the 50-kDa form (glucokinase) survived in mammals but was lost through a mutation event in certain vertebrate families, such as Aues and others. The recent cloning of the rat (Schwab and Wilson, 1989) and human (Nishi et aZ., 1988) HKl as well as rat HK2 (Thelen and Wilson, 1991) and HK3 (Schwab and Wilson, 1991) has provided evidence in support of this gene duplication-fusion hypothesis. Data from these cDNAs indicate that there is significant sequence identity between the mammalian forms, as well as strong similarity to the yeast HKs (Kopetzki et aZ., 1985; Frohlich et al., 1985). Rat glucokinase was also found to be highly similar to both the 3’ catalytic half of HKl and to the yeast isozymes (Andreone et al., 1989). Here we report the cloning of the bovine brain HKl using knowledge of the evolutionary conservation of HK amino acid and nucleotide sequence. This has allowed us to determine the overall alignment of the HK and glucokinase sequences and will facilitate the cloning and sequencing of HK and related genes in other species. We have identified regions corresponding to important functional domains in both halves of the HK cDNAs, lending further support for the evolutionary origin of mammalian HK l-3 by duplication-fusion events. We discuss the amino acid sequence alterations in the regulatory and catalytic domains and their effects on functional properties of the enzyme. We construct a phylogenetic tree for the HK

We have amplified and sequencedthe complete coding region of bovine hexokinase isoenzyme 1 (IIKl) from brain RNA with PCR primers selected for sequenceconservation. The sequenceinformation was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes. Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HKl outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of IIKl essentialto enzyme function. The ATP-binding site in the catalytic half of the IIKl protein resembles nucleotide-binding regions from protein kinases,with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the lossof HKl catalytic function in the regulatory domain. Sequencecomparisonssuggestthat the SOkDa mammalian and yeast glueokinasesarose separately in evolution. In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencingof the coding regions for other HKs and related proteins. o 1991

o&3&7543/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

AND

of Pediatrics, *Molecular Biology Information Resource, Baylor College of and Biophysical Sciences, University of Houston, Houston, Texas 77030 ReceivedJune

Academic

V. ADAMS,*

MAMMALIAN

HEXOKINASE

1: EVOLUTIONARY

Coding Region

5’ UTR

1015

CONSERVATION

3’ UTR +-I

l-l-

2762 AA-100% N-957.

RR-loo% N-1007.

2760

I=

RiO%

AA-100%

N-1007. dT -

91-30 iizrO7. N-1007. 1% MOPRC

RG%

tlfl-100% N-1007.

N-1007. 23

RR-100%

N-92% 1159 liiOO% N-92%

2761 flciYO% N-1007.

dT -

N-927.

ys MOPRC

FIG. 1. HKl cloning strategy. Four primer pairs were used to amplify the coding portion of the bovine HKl cDNA. Percentages under each primer indicate the amino acid and nucleotide conservation between rat and human HKls. Arrows represent the direction from which amplification occurs. Right arrows (+) are sense strand primers. Left arrows (+) represent antisense primers. Untranslated regions (UTR) were generated with one nonspecific oligo(dT) primer [dT], and one specific primer near the end of the coding regions.

family and propose for the first time a novel evolutionary origin for mammalian glucokinase.

nucleotide Biosystems sizer.

mixtures were synthesized on an Applied 380B or a 392 oligonucleotide synthe-

METHODS

Template

Materials

Tuq polymerase was obtained from Perkin-ElmerCetus. Reverse transcriptase (Moloney murine leukemia virus, MoMLV) was purchased from Pharmacia. Bovine brain was obtained from Texas A&M University Department of Animal Sciences, and freeze clamped (Lowry and Passonneau, 1972) within 2 min of death using aluminum blocks cooled in liquid nitrogen. Sequenase (version 2.0) and dideoxynucleotides were obtained from United States Biochemicals. Primer

Design and Synthesis

The rat brain and human kidney HKl cDNA sequences were compared to identify short (25-30 bp) regions of 100% sequence identity. Primers were designed to span 500- to 900-bp regions of the bovine HKl cDNA, and were constructed as unique oligonucleotides. Four pairs of primers were used to amplify overlapping coding portions of bovine HK (Fig. 1). Primers corresponding to regions with nucleotide homology greater than 88% were considered acceptable for use based on previous results with primers of high degeneracy or mixed oligonucleotide primed amplification of cDNA (MOPAC, Griffin et al., 1989). Untranslated regions were amplified using one specific internal primer and a nonspecific primer for the other end (Frohman et al., 1988). The nonspecific primer consisted of a poly(dT) stretch with an attached linker. The linker contained three unique restriction enzyme sites including CZuI, XhoI, and SalI. The oligo-

Preparation

and PCR

Total RNA was isolated from bovine brain using the RNAzol method first described by Chomczynski and Sacchi (1987) and was then used for first-strand cDNA synthesis. First-strand cDNA for each reaction was generated from 1 pg of total RNA with MoMLV reverse transcriptase and either an oligo(dT) primer or the 3’ primer of each primer pair (Nos. 91-30,2758, 2761, 1160, or 2762). Each of the 3’ primers corresponded to the antisense strand. In order to amplify the 5’ untranslated region it was necessary to add a poly(dA) tail onto the first strand cDNA using terminal transferase and dATP. The nonspecific primer could then be used in the amplification of this portion of the cDNA. A total of 5 pmol of primer was added to 1 kg of total RNA, in the presence of 20 U of an RNase inhibitor, RNasin. The mixture was heated to 95°C for 5 min to remove RNA secondary structure and immediately cooled on ice. To each first strand reaction was then added 10 ~1 2 mM dNTPs, 2 ~1 of 10X PCR buffer (10X: 200 mM Tris, ph 8.3,500 mhf KCl, and 25 mM MgCl,), 20 U RNasin, 200 U of MoMLV reverse transcriptase, and DEPC-treated water to 20 ~1. Each reaction was allowed to proceed for 1 h at the appropriate empirically determined temperature (37,48, or 55“C). Due to instability of the reverse transcriptase at temperatures higher than 37°C additional enzyme was added more frequently to those reactions requiring the two higher incubation temperatures. Each tube was then heated to 95°C to inactivate the en-

1016

GRIFFIN

zyme. A total of 45 pmol of additional primer 1 (used in the first reaction), 50 pmol of the second primer, and 8 ~1 of reaction buffer were added to the appropriate reaction mixtures and then were diluted to 100 ,ul total volume with water. Taq, 2.5 U, was added after an initial 5-min incubation at 94’C. Thirty rounds of amplification were performed at the following initial conditions: 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min using a Perkin-Elmer-Cetus Thermocycler. Ten microliters of each reaction was analyzed on a 2% ME agarose gel in 1 X TBE (90 mMTris/64.6 mM boric acid/2.5 mM EDTA, pH 8.0). Those primer sets that generated multiple product bands were then amplified using higher annealing temperatures, while several failed initially to generate products of the correct size. Alteration of conditions for the first strand cDNA synthesis, specifically, increased temperature for reverse transcription, eliminated the problem of incorrect size. Higher annealing temperatures for the PCR failed in most cases to reduce the number of products. Hybridization of these products to rat HKl not only identified the correctly sized products as bovine HK, but also indicated that several of the others were related to HK. It was noted during these experiments that the crucial step for generating full-length products was production of the first strand cDNA and not PCR. Gels were denatured in 0.4 N NaOH and transferred to a Zetaprobe nylon membrane overnight in 0.4 N NaOH by Southern blotting (Maniatis et al., 1982). A rat HKl cDNA was random hexamer labeled with [a3’-P]dCTP (Amersham) and Klenow fragment of DNA polymerase (Feinberg and Vogelstein, 1983). Filters were prehybridized (Church and Gilbert, 1984) then hybridized in Church buffer at 68°C with 2 X lo5 cpm/ml of labeled probe for 12-18 h, and washed in 0.4 M sodium phosphate, 0.1% SDS, at 65°C for 1 h, followed by 0.1X SSC, 0.1% SDS at 75°C for 15 min. Filters were exposed to Kodak X-OMat film at room temperature. The filter was then stripped and rehybridized to a 45-bp oligonucleotide primer, which corresponded to the internal region of the original MOPAC product (Griffin et al., 1989), under the same conditions. Appropriate products were reamplified for use as template for sequencing or as template for asymmetric PCR.

DNA Sequencing Products of the correct size were sequenced directly after reamplification using asymmetric PCR to decrease the problem of reading errors when amplified products are subcloned and sequenced. Using the primer pairs designated above, reaction mixtures containing 50 pmol of one primer and 1 pmol of the other

ET

AL.

were amplified and sequenced by the asymmetric PCR method (Gyllensten and Erlich, 1988) or using the direct method of Kusukawa et al. (1990). Reaction mixtures were as described above (10X buffer: 500 n&f KCl, 100 mM Tris, pH 8.3,15 mA4 MgCl,). Reaction mixtures were applied after PCR to a Centricon30 microconcentrator (Amicon) to remove the excess dNTPs and buffer components. The columns were washed three times with water, and spun at 4000g. Retentates of 35 ~1 were collected for each; 3-7 ~1 (1 pmol) of each was added to 10 pmol of the appropriate primer in 1X sequenase buffer. Annealing of the single strand was performed at 65°C for 15 min, followed by room temp incubation for 15 min. The sequencing reactions then proceeded as per manufacturer’s specifications (United States Biochemicals).

Sequence Alignment

and Phylogenetic

Analysis

N- and C-terminal halves of the vertebrate HKs were analyzed independently. The boundaries of duplication were defined by dot-matrix homology plot; nonduplicated portions were trimmed away. The separate homologous regions of the vertebrate HKs were aligned as monomeric units. Protein sequences were aligned using the PIMA (Pattern Induced Multiple Alignment) program (Smith and Smith, 1990). The aligned protein sequences were used as a guide to align the corresponding DNA sequences using the program PIMA-to-paup. Phylogenetic trees were constructed based on the aligned DNA sequences. Programs in PHYLIP (Phylogeny Interference Package version 3.3; J. Felsenstein, Department of Genetics, University of Washington, Seattle, WA) were used for creating phylogenetic trees. The program DNADIST generated a matrix of genetic distances for all pairwise combinations of nucleotide sequences in the alignment. Distances were corrected for nucleotide reversions by the two parameter model of Kimura (1981). The distance matrix was analyzed by the program KITSCH to construct the phylogenetic tree by the Fitch-Margoliash least-squares method (Fitch and Margoliash, 1969). RESULTS Six fragments, which represented the complete coding region of the bovine HKl cDNA, were successfully amplified by PCR from bovine brain RNA. The sequence from these overlapping PCR fragments was compared to all available eukaryotic HK sequences at both the nucleotide and amino acid levels, and the results are shown in Table 1. The sequence similarity ranges from approximately 90% among the other mammalian HKls to 49% when the bovine HKl cod-

MAMMALIAN

HEXOKINASE

1: EVOLUTIONARY

TABLE Homologies” mhklc mushkl-cc rathkl-cd humhkl-c’ bovhkl-c ratgk-1’ mushkl-n rathkl-n humhkl-n bovhkl-n yschka8 yschkb’ yschkc i a Numbers sequences). ’ Numbers phylogenetic ’ GenBank d Ref. (37). = Ref. (35). ’ GenBank 8 GenBank h GenBank i Ref. (1).

0.0000 6.5 13.4 12.5 40.2 41.6 41.3 40.7 42.8 53.5 54.4 54.4 below

rhklc

hhklc

bhklc

0.0696 0.0000 13.1 11.4 39.6 41.3 41.0 40.9 41.2 53.4 54.0 53.5

0.1418 0.1275 0.0000 8.5 39.3 41.2 41.2 39.7 42.1 53.6 55.2 54.1

0.1511 0.1472 0.0923 0.0000 40.3 41.3 40.9 39.7 42.1 53.4 55.4 53.5

the diagonal

indicate

above the diagonal represent tree is based. accession No. JO5277 (Ref.

accession accession accession

No. 504218, No. X03482, No. X03483,

the number the corrected

0.6799 0.6673 0.6876 0.6580 0.0000 41.4 41.7 41.8 43.0 54.8 56.2 57.6 of nucleotide “distance”

Distances*

mhkln

rhkln

hhkln

bhkln

0.7184 0.7132 0.7175 0.7163 0.7313 0.0000 6.2 13.1 16.9 56.8 57.5 57.4

0.7139 0.7071 0.7079 0.7234 0.7362 0.0599 0.0000 13.3 16.1 56.5 57.9 56.9

0.6914 0.6899 0.6853 0.6721 0.7332 0.1434 0.1453 0.0000 11.7 56.8 57.2 56.8

0.7592 0.7555 0.7686 0.7573 0.7892 0.1915 0.1815 0.1278 0.0000 57.7 57.5 59.1

changes between

per 100 bases (the percentage the compared

sequences,

ysha

yshb

yshc

1.3207 1.2773 1.3066 1.2987 1.2964 1.4061 1.3683 1.4246 1.5108 0.0000 23.6 49.1

1.3653 1.3068 1.3892 1.3991 1.4278 1.4276 1.4467 1.4346 1.4964 0.2856 0.0000 50.3

1.4064 1.3242 1.3676 1.3451 1.7016 1.6364 1.5879 1.5629 1.6983 1.0118 1.0602 0.0000

difference

between

the compared

and are the raw data from

which

the

(4)).

Ref. (3). Ref. (30). Ref. (13).

sequence is compared to rat HK3. The higher percentage similarity of the bovine sequence and the other sequences that have been designated as HKl confirms their collective identity as HKl. As in other mammalian HKs, the bovine HK amino half is a duplication of the carboxy half. The two halves of the coding sequence from the bovine cDNA were compared and found to be 58% conserved at the nucleotide and 49% at the amino acid level. All available HK sequences were aligned using the PIMA and PIMA-to-paup programs (Smith and Smith, 1990). Several regions of greater similarity were noted within the aligned sequences (Fig. 2), including the ATP-binding (box B) and glucose-binding (box C) sites. Boxes D and E in Fig. 2 represent other residues that are potentially involved in ATP binding. In addition, we noted that a l&amino-acid N-terminal domain (box A) was absolutely conserved throughout the mammalian HKls at the amino acid level and 92-96% conserved at the nucleotide level. Five amino acids at positions 178, 231, 232, 303, and 338 in the aligned sequences (marked by a +), which are involved in glucose binding in yeast (Bennett and Steitz, 1980), are conserved in all existing HKs and are present in both the amino and carboxy halves of the mammalian HKs. We were able to identify the regions thought to be involved in ATP- and glucose-binding in both halves ing

1

and Phylogenetic rgkl

1017

CONSERVATION

of the protein, although the sequences were slightly altered in the amino half. These regions are illustrated in Figs. 3 and 4. Core regions of conservation were noted for the substrate- and nucleotide-binding sites. Consensus sequences derived for each sits were compared to all peptide sequences in the PIR protein database (George et al., 1986) and failed to recognize any other carbohydrate kinase. We also found that two amino acid positions in the substrate-binding site, and seven amino acid positions in the nucleotidebinding site have residues that are present in the catalytic sites but not in the regulatory (amino half) sites. A lysine residue present lo-13 residues downstream of the ATP-binding site (box B) of the catalytic half of bovine HKl was noted to be altered to a glutamate in this site in the amino half. The evolutionary relationships between the HKs were explored through the construction of a phylogenetic tree (Fig. 5). Nonduplicated regions, including sequences that encode the N-terminal outer mitochondrial membrane-binding domain, were omitted to achieve the best alignment. We used the KITSCH algorithm of Felsenstein to produce a rooted tree with contemporaneous tips, for clarity. The sequences fell into three clusters: yeast, N-terminal, and C-terminal. The yeast HKs were most diverged from the others, while the C-terminal sequences were most conserved as indicated by the depth of the branch bi-

1018

GRIFFIN

rn”ShkI-n rdChk1-n humhkl-n b.¶Yhkl~” !ll”Shkl-C rathkl-c humhkl-c bO”hkl-c ratg)r-1 yschka yschkb yxhkc

----------------------------------------------------------------------MVHLGPKKPQARKGS MWLGPKKPQARKGS -------------MS

91

B

105

rIALDL008SF1 VIUab001SFI

31 45 46 60 61 75 76 90 IDKYLYAMRLSDEIL 1DILTRFKKE"KNOL -SDRYNPI----ASV KIILCTF"RSI8DOSL IDKYLYAMRLSDEIL IDILTRFKKEHKNOL -SDRYNPT----AS" KllLITLLRSIVDaS~ IDKYLYAMRLSDETL IDIMTRFRKEMKNOL -SDRFNPT----AT" KIL?TFVRSI?DOS~ IDKYLYAMRLSDETL wrk.94RfKm.mNar. -sRDFNPT----*TV KILVTFVRSIVDOSL ----------EQHRQ IEETLSHFRLSKQAL MEVKKKLRSEMEMOL -RKETNSR----AT" K,,L?SYVRSI?DOTIC ----------EQHRQ IEETLRHFRLSKQTL MEVKKRLRTEMEMOL -RKETNSK----AT" K,,L?SFVRSI?DaTL ----------EQHRQ IEETLRHFHLTKDML LEVKKRMRAEHELOL -RKQTHNN----A"" K,,L?SFYRRT?DOTL ----------EQHRQ IEETLAHFRLSKQTL MEVKKRLRTEMEMOL -KKf.TNSN----AT" NNL?SFLRSI,DOTI MAMDTTRCGRQLLTL VEQILAEFQLQEEDL KKVnSRnQKE,,DROL -RLETHEE----AS" K"LITY"RST,EOSL -"PK----EWE IHQLEDMFTVPTETL RKWKHFIDELNKOL --TKKGVN------I P,,I8GWVNEF8TOKL "ADVPK----ELMQQ IEIFEKIFTVPTETL QAVTKHFISELEKOL --SKKGVN------I p"I,GWMDF,TaKX FDDLHKATERAVIQA VDQICDDFEVTPEKL DELTAYFIEQMEKOL APPKEGHTLASDKGL P~IVMVTGSVNOTZ 106 ILR+N"EK--SON ILK ILK

rrhmmwsfn

120

121 135 136 150 VSMESLVYDTPENIV --HGSGSQLFDH-"A

ILR

IRSGK--KRT ru&&aaTNFn

mushkl-n rathkl-n humhkl-n bcvhkl-n mushkl-c rathkl-c humhkl-c bovhkl-c ratgkLl yschka yschkb yschkc

QQSKIDEAILITIT RQSKIDEAILITVT

271 CYNEELRHI-----D CYMEELRHI-----D CYMEELRHI-----D CYMEELRQI-----D

""shkl-n rathkl-n humtikl-n bovhkl-n mushkl-c rathkl-c humhkl-c txwhkl-c rargk-1 yschka y.,chkb yschkc

I K

300

60 60 60 70 7s 7s 77

151 151

VHMESCVYDTPENIV VHMES~YYDTPENIM VEMHNXIYSIPLEIH VEMHNKIYSIPLEIM YEMHNaIYAIPIEIM VEMHNIIYSIPIEIM

--HGSGSQLFDH-“A

E----cLGDFMEKRK

157

--"GSGSQLFDH-VL

E----CLGDFHEKKK S----CISDFLDYNG

141

--QGTGDELFDH-I”

157

--WGTGDELFDH-IV

S----CISDfLDYMG

--QGTGDELFbH-I”

S----CISDFLDYHG

141

--QGTGEELFDH-I"

S----CISDFLDYHG

141

--TGTAEMLFDY-IS

E----CISDFLDKHQ

153

L----KDFM"EQELL L----KAFIDEQFPQ

160

IKGP

141

160

KDRKPMt(LarTrlYVl

LRFHKKYHPDELAKG

166

l

210 211 225 226 ** ASGVEGADWK LLNKAIKKRGDYDRN 1VA~TVGTbK-W ASGVEGADVVK LLNKAIKKRGDYDAN IVImTVGT"MTC ASGVEGADWK LLNKAIKKRGDYDAN IVA~TVGT"MTC

286

60

D----cLGDFPIEKKK

240

241

255

256

D

270

GYDD-----------

QQCEVOLII~~~

236

GIDD----------GYDD-----------

QQcE\nLI QHCEVQLI

236 236

LLDKAIKKRGDYDAN

IVaVVIPTVGTMIDC

GYDD-----------

QHCEVOLI

236

LLRDAVKRREEFDLD LLP.DAVKRREEFDLD LLRDAIKRREEFDLD LLRDAVKRREEFDLD LLRDAIKRRGDFEMD LMKEISKRELP-IE MLQKQIsKKNIP-Is LYQEQLSRQGMPMIK

W~W~PTVGTMMTC

AXEE----------AYEE----------A~EE----------AYEE----------YYED----------YYTD----------YXTD-----------

PSCEIOLZ PTCEIOLI PTCEVOL~ PTCEVOL RQCE"OU PETKMOV PETKMOV SEP"IaC

220

301*

LVEGD----EGRRCI LVEGD----EGRs(CI LVEGD----EGR*CI LGWGD----DGR#CI

PTIWQRVGDDGSLED

CYMEEMKNV-----E

315

W~W~~PTVGTMMTC WA~TVGTHMTC

W~~W~PTVGTHHTC WIMV~PTVATMISC IVILImTVGTLIRS WALImTTGTLVAS WALTIPTVGTYLS" 316

CTTSDNTDSMTSGEI

330

331

*

345

220 220 220 232 238 238 256

346

360

PTEWODTGDDGSLED

IRTEFDREIDRG-BL IRKEFDREFRRG-IL

NPOK~LF~VSOMY NPOKOLF~SOMY NPaKoRFmSaKY

MVEGN----QGQmCI

WawOArGDNGCLDD

IRTDFDKVVDEY-no

NSOK~Rf~ISOMy

MGELVILILVKMAKE MGELVILILY-KS LGELVULILVFAMKE MEDWIL"LW,%F,KE LGEIVUIILIDFTKK

CYMEEHKNV-----E

MVEGN----QGQYcI

MIWOAIGDNGCLDD

IRTDFDKWDEY-IL

NsOKORF&~ISOHY

LGEIvWILIDFTKK

300

CYMEEHKNY-----E

MVEGD----QGQWCI MVEGN----QF@CI

IRTHYDRLVNEY-8L IRTDFDKVVDEY-IL

NROKORY~KMISOMY NSONORF&KMISOIY

CYHEEMQNV-----E AFYDVCSDIEKLEGK AYYDYCSDIEKLQGK

LVEGD----EGPMCV LADDI--PSNSPmRI LSDDI--PPSRPs(AI LRDKLIKEGKTH~II

FLLEYDWWDES-IA

NPOPOLYSXIIGOKY

PRTKYDVAVDEQ-BP PRTKYDITIDEE-BP PTTKYDVVIDOKLIT

RP~,,F~TSaYY RPQPOTF‘X"SSOYY NPOFHLF~IRVSOMF

LGEIVWILIDFTKK LGEIVINILIDFTKK MGELVILVLLKLVDE LGEILRLVLLELNEK LGEII&AL~~DHYKQ LGEVLINILVDLHSQ

300

CYMEEHKNV-----E

UNaArGDNGCLDD *MNOAVGDNGCSDD NTINOATGDSGELDE HCfiYaSrIaNEHLVL PC&YaSr-DNEHWL IVENaSV-aNELK"L

CYHEEINKITKLPQE 361

““shkl-n rathkl-n hmhkl-n bovhkl-n mushkl-c rathkl-c humhkl-c bovhkl-c ratqk-l yschka yschkb

285

196 X

76

--HGSGTQLFDH-VA

l

181 c 195 RQSKIDEAVLITIT

76

151

TQSKYKLPHDMRTTK --"QEELWSFIA-DS TQSKYRLPDAMRTTQ --NPDELNEFIA-DS EQMISKIPDDLLDDE NVTSDDLFGFLARRT

LGGDR-TFDT LHGDH-TFDT

76 76

VSMESLIYDTPENIV

“KTIHQMYSIPEDAM

yschka yschkb yschkc

rathkl-n hwnhkl-n bovhkl-n mushkl-c rathkl-c htikl-c bovhkl-c ratgk-1 vschka yschkb yschkc

AL.

30 WKK QVKK QVKK PVKK

Kl”Shk1-n rShkl_” h”*kl~n bO”hkl-” lll”Shkl-C rathkl-c humhkl-c bO”hkl-c catgk-1

m”Jhk1-n

ET

375

SLLF-----EGRITP GLLF-----EGRITP GLLF-----EGRITP GLLF-----EGRITP GFLF-----RGQISE

376

IRTEfDRELDRG4L

IITIwQ~GDDGSLED

390

391

405

ELLTRGKFTTSDVAA ELLTRGKFNTSDVSA ELLTRGKFNTSDSVR ELLTRGKFNTSDVSA PLKTRGIFETKFLSQ PLKTRGIFETKFLSQ TMKTRGIFETKFLSQ PLKTRGIFETKFLSQ QLRTRGAFETRFVSQ KLKQPyIHoTSyPAR KFDKPFVPLDTSYPAR

IBDPFENLEDTDDH

GLLLQQYRSKEQLPR

"LTTPFQLSSE"LSH

ICIDDSTGLRETELS

451 465 NKGTPRLRTTVGVDO NKGTPSLRTTVGVDO NKGTPdLRTTVGVo@ NKGTPRLRTTVGVDO NRGLDHLNVTVGVDO NRGLDHLNVTVGMQ NRGWRLNYTVGVDO NRGLDRL.NVTVG"Da SRSEDVMRITVGVDO CQKRGYKTGHIRAOO CQKRGYKTGHIAADO

466

LNKRYHGEVEIGCDO

SWEYYIGFRSHLRH

GFLF-----RGQISE

GFLF-----RGQISE GFLF-----RGQISE NLLF-----"GEASE GLML-----KDQDLS GFIF-----KNQDLS

NPQKQLF~XWSOMY

~YT&!GW.~GDDGSLED IRTEFDRELDRG-IL

480 SLYKM"?QYSRRFHK SLYKMHVQYSRRFHK SLYKTHVQYSRRFHK SLYKT"?QYSRRF"K TLYKLHIHFSRIMHQ TLYKLH,“FSRIMHO TLYKLH,“FSRI,4”P TLYKLH,QFSRI""Q S"YKLH?SFKERF"A S"YKLH.GFKEAAAK SVYNKYIGFKEKAlVl

406

420

IETD--KEGVQNAKE IIKD--KEGIQNAKE IIKN--KEGIQNAKE ILKD--KEGL"NAKE

ILTRLGVEPSHDDC" ILTRLG"EPSKD"C" ILTRLGVEPSDDDC" ILTRLGvERSDDDCY

ILSD--KLALLQvRA

~L~QLGLNSTCSDSI

IISD--RLALLQVRA ILSD--RLALLQVRA IISD--RLALLQVRA VISD--SGDRKQI"K ICDDPFENLEDTDDM

ILQQLGLNSTCDDSI ILQQLGLNSTCDDSI ILQOLGLNSTCDDSI ILSTLGLRPSVTDCD FQKDFGVKTTKPERK FQNEFGINTTVQERK LLQSLRLPTTPTERV

495 481 TL-------RRLVPD TL-------RRWPD TL-------RRLYPD TL-------RRL"PD TV-------KELSPK TV-------KELSPK TV-------KELSPK TV-------KELSPK SV-------RRLTPN GLRDIyGWTGENASK ALKDIYGNTQTSLDD AL-ALSPLGAEGERK

496

SDYRFLLSE SDVRFLLSE SDVRFLLSE SDVRFLLSE CTYSFLLSE CTVSFL CN"SFL CNVSFL CEITFI DPITI" YPIKIYPAE YHLKI--A

E

510

421

435

SVQHLCTIVSFRSAN SVQHICTIVSFRSAN S"Q""CTI"SF.SAN SVQHVCTIVSF~SAN LVKTVCGWSKMQ LVKTYCGWSK~Q

436

316 316 316 300

300 312 324 324 345

450

LVAATLGAILNRLRD LVRATLGAILNRLRD LVMTLGAILNRLRD LVAATLGAILNRLRD

399 399 399 399

LCGAGMAAWEKIRE

383

LCGAG&AA""EKIRE LCGAGMAAWDKIRE LVKTVCGWSKIVIPQ LCGAGMAAWEKIRE IVRRI\CESVST~H.HCSAGLAGVINRILRE LIRRLCELIGTtiR LA"CGIMI-----LIRRLSELIGALAAR LSYCGIMI-----QIQKLVRAISRISAY LAAVPLAAILIKTNA

393

LVKTVCGWSRURQ

511

383 383 385 403 403 435

525 +I AV$&yRLA--&"AYRLA--A"*yRL*--I\"AYLM---

-----------------------------

AVGVRLRGDP

TNA-----

455

SIA----SS-----AIS----AQ-----KVSGIIGA KSVGIIGA -----___

455

ITAVGYRLRGDP ITAVGVRLRTEA ITAYGVRLRGES “SAYACKKAC”L IMLSEKRIAEG IMLAQKRIAEG .?&tJA-------

316

464 464 464 464

454 455 466 487 487 501

MAMMALIAN

HEXOKINASE

1: EVOLUTIONARY

1019

CONSERVATION

Boulne HK I -n Rat HKI -n Human HKl -n Mouse HKI-n Bouine HK I -c Rat HK I -c Human HKI -c

ITWTK

Rat HK4 Yeast HKPI Yeast HKPI I Yeast 6lk

GFTFSF GFTFSF GFTFSF

VRHEDL

+ Consensus:

P*GFTFSFP..Q..*

*.*.L.

. WTK

FIG. 3. Substrate-binding site of the eukaryotic hexokinases. Both regulatory and catalytic halves of the HKls as well as rat (glucokinase) and the yeast hexokinases are shown. Residues that are conserved in two-thirds of the sequences are boxed. Two positions have an amino acid uniaue to the catalvtic site are marked with a +. A consensus sequence is indicated. Asterisks (*) indicate positions conservative or semiconservative amino acid changes.

furcations within each group. The rat glucokinase was most closely related to the C-terminal halves of the HKs, whereas the yeast glucokinase was most closely related to the yeast HKs. This observation suggests that the duplication leading to rat glucokinase was a separate (and more recent) event from the duplication leading to the yeast glucokinase, implying that the glucokinase activities arose twice in evolution. DISCUSSION

We have shown that the bovine brain HKl cDNA exhibits significant homology both to other mammalian HKs and to the yeast 50 kDa HKs. Our sequence data, along with that from other groups (Schwab and Wilson, 1989; Nishi et al., 1988), support the earlier hypothesis that HKl arose as a result of a gene duplication and fusion of an ancestral HK protoenzyme (Ureta, 1982). This was followed by functional divergence of the duplicated halves creating the regulatory and catalytic domains of HKl (Fig. 6). Comparisons of the bovine coding sequence to that of other HKs allow us to identify not only the functional binding domains of HKl but also to pinpoint key residues within those domains. We have shown that essential

HK4 that with

domains are conserved in each of the duplicated halves of HK. Alignment of the eukaryotic HKs allowed for both the identification of highly conserved coding regions (discussed below) and for the generation of a phylogenetic tree. Several interesting conclusions could be drawn from the tree. Most notably, it appears that the 50-kDa mammalian and yeast glucokinases do not have a direct common ancestor, suggesting that the gene-encoding glucokinase (HK4) arose at least twice during evolution. We determined that the mammalian glucokinase (HK4) may have arisen after the duplication-fusion-reduction event that created the mammalian HKs, by loss of the N-terminal half, suggesting that it was a more recent evolutionary event. This would perhaps explain why some nonmammalian vertebrate species lack a glucokinase isozyme (Ureta, 1975). In addition, we noted that a much more ancient event gave rise to the yeast glucokinase isozyme presumably from the HK protoenzyme. Consistent with the duplication-fusion-reduction hypothesis is the observation that the rat glucokinase enzyme is more closely related to the C-terminal half of mammalian HKl than to the N-terminal half. It has been noted that in rat liver, glucokinase interacts with a

FIG. 2. Alignment of the eukaryotic hexokinases. Available hexokinases, including the hepatic form of glucokinase (rat gkl), were aligned. Residues that are conserved in all existing hexokinases are offset. Residues of unknown function are located outside the boxed regions. Amino acid positions thought to be glucose contact points are marked by a l . An asterisk (*) marks the position that corresponds to a critical lysine residue (18) in the ATP-binding site of the protein kinases. Box A, outer mitochondrial membrane-binding domain; B, ATP-binding site; C, glucose-binding site. Boxes D and E represent alternate ATP-binding sequences (G-X-G-X-X-G/A), which may function in the binding process (Refs. (22,23,35)). Right angle arrows (CI, r) demark portions of the sequence used in phylogenetic analysis. Numbers to the right indicate the position within the sequence; numbers on top indicate the position within the alignment. Species and sequence identification are to the left. Top four sequences (ending in “-n”) are N-terminal half; those ending in “z” are the corresponding C-terminal sequences. The N-terminal halves are thought to have regulatory function (Ref. (48)). All other sequences possess catalytic activity. yschka, yeast HK PL yschkb, yeast HK PII; yschkc, glucokinase; mushkl, mouse HKl.

1020

GRIFFIN

Protein

AL.

M-U-l

loulne HKl -n Rat HKl-n Human HKI -n Mouse Boulne Rat HKI Human Mouse Rat HK4

ET

F

HKl -n HKl -c -c HKI -c HKI -c

R I R V R V V R V R VM l

Kinases:

CAMP-dep cGMP-dep

.

F . F . F . F. F. F. F . F .

PK PK

L L L L L L L L

R R R R L L L L L

V V V V V

Q...+13....E Q...+13....E Q...+13....E Q...+13....&

V

. ..+13... K...+13...

V V V

K K

K K

K...+13...

. ..+13...

K

*

F G R V M L V F G R V E L V

iM

K...+13...

l

. ..+ll... Q...+12...

i

K K

FIG. 4. Nucleotide-binding site of the mammalian hexokinases. Regulatory (“-n”) and catalytic (“-c”) domain sequences are compared to the ATP-binding site sequences of the bovine CAMP-dependent and cGMP-dependent protein kinases. Residues that are conserved are boxed. Positions that have an amino acid unique to the HKl catalytic site are marked with a +. A Iysine residue, thought to play a key role in the phosphoryl transfer of the y-phosphate of ATP, is present 11-13 residues downstream of the main binding site of the protein kinases, rat HK4, and the catalytic domain of the HKls, but not in the regulatory half.

regulatory protein (Vandercammen and Van Schaftingen, 1990) and it has been postulated that this regulatory protein actually corresponds to the N-terminal regulatory half of HK (Schwab and Wilson, 1991). Taken together this information suggests that glucokinase arose after the evolution of the N-terminal half of the HKs into a regulatory domain and that after the reduction event, the regulatory domain may have been retained as an independent gene product (Fig. 6).

7

yschkb

I

yschka yschkc bovhkl

n

humhkl

n

rathkl

n

mushkl ratgk

n 1

humhkl

c

bovhkl

c

rathkl

c

mushkl

c

FIG. 5. Phylogenetic tree for the hexokinases. A rooted tree was produced using the KITSCH algorithm of Felgenstein (Phylogeny Interference Package version 3.3, University of Washington, Seattle). Tree constructed for the mammalian HKls, mammalian glucokinase, and all yeast hexokinases. Those sequences ending in “-n” are N-terminal half; those ending in “-c” are the corresponding C-terminal sequences. yschka, yeast HK PI; yschkb, yeast HK PII; yschkc, glucokinase; mushkl, mouse HKl; bovhkl, bovine HKl; humhkl, human HKl; ratgk-1 = hepatic form of rat HK4 (glucokinase).

Putative ATP- and glucose-binding domains were identified in the carboxy-terminal (catalytic) half of bovine HKl by alignment and comparison to other HKs and other proteins that possessATP-binding regions. Inspection of the corresponding regions in the amino (regulatory) half of the bovine protein showed that these same domains were present. The substrate-binding site was slightly more divergent in the regulatory half, but a core of conserved sequences could be identified. Our results indicate that these alterations in nonessential residues in the primary structure may allow for changes in specificities, although we cannot rule out changes in other residues involved in the secondary and tertiary structures that may alter the affinities of these domains. The binding site in bovine HKl is very similar to the putative ATP-binding domains of several protein kinases (Zoller et al., 1981; Hashimoto et al., 1982; Hanks et al., 1988), oncogenes such as c-myc (Hanks et al., 1988) and c-src (Kamps et al., 19&M),and several other ATPases. There have been other nucleotide binding sequences reported in proteins such as ATP synthase (Walker et al., 1982), adenylate kinase (Fry et al., 1986), and Fl ATPase (Cross et al., 1987). Notably, the mammalian and yeast HKs possess the former, but not the latter types of binding sequences. In addition, this HKl site does not have recognizable similarity to other carbohydrate kinases. A portion of another ATP-binding domain of a bovine 70 kDa heat shock cognate protein (Flaherty et al., 1990) has also been noted to be conserved in the HKs. We find the protein kinase ATP-binding core sequence in all HKs examined, including rat and yeast glucokinase (Andreone et al., 1989; Albig and Entian, 1988). Of note, all of the catalytically functional ATP-binding domains of both the protein kinases and HK have an invariant lysine that is found lo-15 residues down-

MAMMALIAN -HK

1: EVOLUTIONARY

Protoenzyme

cat.

/

HEXOKINASE

\

GENE DUPLICATION FUSION

AND

/ \

-aYeast Yeast

HKPI, PII. & glucoklnase

cat. Alt.

cat.

I

Mammalian HK4 (Glucoklnase) Deletion gene to create

of S’half glucoklnase

ISOZYME EVOLUTION

of \

FIG. 6. Evolution of the eukaryotic hexokinases. An evolutionary model is proposed for the eukaryotic hexokinases based upon phylogenetic analyses as well as structure-function considerations. The yeast isozymes evolve separately from the events that will give rise to the vertebrate and mammalian isozymes. The 100kDa mammalian hexokinases would have been created from a gene duplication and fusion event, followed by evolution of one of the halves into a regulatory domain. The mammalian glucokinase may have arisen either before (alt. 1) or after (alt. 2) the evolution of the regulatory domain through loss of the 5’ half of the gene. Exon recruitment may be responsible for the addition of an outer mitochondrial membrane porin binding domain (PBD) to HKl and HK2.

stream (i.e., toward the C-terminus) of the core sequence (Hanks et al., 1988). It is also intriguing that at least one of the yeast HK isozymes (HK PII) has been noted to have protein kinase activity (Herrero et aZ., 1989) and recently we have been able to show that rat HKl possesses not only protein kinase activity but also the ability to autophosphorylate (Adams et al., manuscript submitted). It has been postulated that this conserved lysine residue is responsible for the interaction with, and phosphoryl transfer of, the y-phosphate of the ATP molecule (Kamps and Sefton, 1986). In yeast HK, this lysine was shown to interact with both trinitrophenyl ATP (Arora et al., 1990h) and PLP-AMP (pyridoxal 5’-diphosphate-5’-adenosine) (Tamura et al., 1988), a potent inhibitor of yeast HK PII, suggesting an es-

CONSERVATION

1021

sential role for this residue in HK catalysis. Mutation of this lysine in oncogenes results in loss of protein kinase activity (Snyder et al., 1985; Hannink and Donoghue, 1985). It is known from crystallographic data that this lysine is positioned on the surface of the small lobe of HK where it can rotate into the active site (Shoham and Steitz, 1980). It is interesting that in the putative ATP-binding domain in the regulatory half, this lysine is replaced by a glutamate residue in bovine, rat, human, and mouse HK. This nonconservative amino acid change may represent the structural basis for the loss of catalytic function at this site, while maintaining its binding properties for adenine nucleotides. Two groups (Schwab and Wilson, 1989; Nishi et al., 1988) have proposed an alternate ATP-binding site, while a third was proposed by Herrero et al. (1989) (boxes D and E, Fig. 2). Each of these sites do possess the consensus sequence Gly-X-Gly-X-X-Gly(Ala) found in the protein kinase domains, but we expect that neither site will represent the correct binding domain because both lack the critical lysine residue. However, as these sites are conserved in both halves of bovine HKl, it may be that they do function in nucleotide binding. The ATP-binding domain that we identify does lack this glycine cluster, which may represent an “elbow” that surrounds and contacts the ribose moiety of the molecule (Sternberg and Taylor, 1984). Differences in substrate specificity and fimction between HK and the protein kinases may necessitate the placement of this glycine cluster in a different spatial position relative to the active site to preserve proper HK function. Certain residues (alignment position: Ser-178, Asn-231, Asp-232, Glu-303, and Glu-338) that were shown to be catalytically important for glucose binding in yeast HK PI and PI1 by X-ray crystallography are present in bovine HKl (Bennett and Steitz, 1980; Anderson et al., 1979; Harrison, 1985). These residues were shown to be present in the 5’ regulatory half as well. One of these residues, Ser-178, is positioned within the putative core glucose-binding sequence Gly-Phe-Thr-Phe-Ser-(Phe/Tyr)-Pro-Cys (bovine HKl AAs G-599 to C-606; alignment Nos. 174-181). This serine presumably is involved in hydrogen binding to the 6-hydroxyl group of glucose, an interaction that is critical for proper conformational changes of the active site, which include rotation of the smaller lobe toward the larger lobe (Anderson et al., 1979). In addition, an important proline residue (Pro-172, alignment) is present in all HKs except yeast glucokinase. This core site was shown by Schirch and Wilson (1987) to be included in a peptide that is labeled by the glucose analog, N-(bromoacetyl)-D-glucosamine, identifying it as the active site.

1022

GRIFFIN

Protein studies (Nemat-Gorgani and Wilson, 1986; White and Wilson, 1987) have provided evidence for the existence of binding sites for glucose and glucose6-phosphate in both halves of the protein. This evidence is supported by studies (White and Wilson, 1989) that indicate that each half, when removed from the other, had the ability to bind to both substrate and inhibitor. These studies have failed to show, however, that the binding domains are separate entities. In fact, the sequence comparisons to other HKs that we report here suggest the presence of only one substrate domain per half, indicating that glucose-6-phosphate binds to the altered substrate site in the amino half. We predict that alterations in certain residues of this domain may allow glucose-6-phosphate to bind with a higher affinity to the site in the amino half and glucose to bind the site in the carboxy half in the native protein. The final domain examined was the putative outer This mitochondrial membrane-binding domain. amino terminal l&amino-acid sequence is predicted, in mammals, to be an LX helical structure based on secondary structure analysis. This mammalian a! helical domain is longer than the N-terminal domain of yeast that is predicted to project out from the rest of the enzyme (Anderson etal., 1979). Sequence comparison between bovine, rat, human, and mouse HKl indicates that these N-terminal 15 amino acids are 100% conserved. Such absolute conservation of this region suggests that it serves an essential function. We have used this information to develop a reporter gene construct coding for a chimeric protein consisting of the N-terminal HKl 15 amino acids coupled to chloramphenicol acetyltransferase (CAT) and have shown that this HKl domain is necessary and sufficient for HKl binding to porin (Gelb et al., manuscript submitted). The family of HK enzymes is responsible for the phosphorylation of glucose, a key control point in carbohydrate metabolism. Since each HK isoenzyme exhibits different tissue distributions and kinetic properties, knowledge of the molecular basis for these properties would be helpful in understanding these enzymes and their detailed cellular roles. Our ability to study HK at a molecular level, and to understand how structural features affect function of the enzyme, has been acquired very recently with the cloning of various HK cDNAs, including bovine brain HKl. Alignment and comparisons of these sequences supports the theory of Ureta and other groups that the mammalian HKs arose from the duplication and fusion of an ancestral protoenzyme and suggests that the yeast and mammalian glucokinases arose twice in evolution. We have described a method for cloning the cDNA for a low abundance protein using knowl-

ET

AL.

edge of the evolutionary conservation of amino acid and nucleotide sequence. This not only permits the isolation of a specific cDNA for a protein that shares common features with many others, it also allows for the rapid identification and sequencing of HKs from other species. In addition, identification of core binding domain sequences will allow for the use of site-directed mutagenesis, and/or reporter gene constructs, to alter key residues and determine the effects of these changes upon HK function. ACKNOWLEDGMENTS This Defects and by Baylor Baylor of Child Health.

work was supported in part by a March of Dimes Birth Foundation Predoctoral Fellowship (18-88-18) to L.D.G. grants (ROl HD22563 to E.R.B. McCabe; P30 HD24064, Mental Retardation Research Center; and P30 HD27823, Child Health Research Center) from the National Institute Health and Human Development, National Institutes of

REFERENCES 1.

ALBIG, W., AND ENTIAN, K-D. (1988). Structure of yeast glucokinase, a strongly diverged specific aldo-hexose-phosphorylating isoenzyme. Gene 73: 141-152.

2. ANDERSON,

C. M., ZUCKER, F. H., AND STEITZ, T. A. (1979). Space-filling models of kinase clefts and conformation changes: Comparison of the surface structures of kinase enzymes implicates closing clefts in their mechanism. Science

204:375-380. 3. ANDREONE, T. L., PRINT, M. A., AND GRANNER, of rat liver glucokinase

R. L., PILKIS, S. J., MAGNIJSON, D. K. (1989). The amino acid sequence deduced from cDNA. J. Bill. C/rem.

264:363-369. 4. ARORA, K. K., FANCIULLI,

5.

6.

7.

8. 9.

10.

M., AND PEDERSEN, P. L. (199Oa). Glucose phosphorylation in tumor cells: Cloning, sequencing, and overexpression in active form of a full length cDNA encoding a mitochondrial bindable form of tumor hexokinase. J. Biol. Chem. 265: 6481-6488. ARORA, K. K., SHENBAGAMURHTI, P., FANCIIJLLI, M., AND PEDERSEN, P. L. (199Ob). Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP. J. Biol. Chem. 265: 5324-5328. BENNETT, W. S., AND STFXIX, T. A. (1980). Structure of a complex between yeast hexokinase A and glucose. II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer. J. Mol. Sol. 140: 211-230. CHOMCZYNSKI, P., AND SACCHI, N. (1987). Single step method of RNA isolation by acid guandinium thiocyanatephenol-chloroform extraction. Anal. Biochem. 162: 156-159. CHURCH, G. M., AND GILBERT, W. (1984). Genomic sequencing. Proc. N&l. Acad. Sci. USA 81: 1991-1995. CROSS, R. L., CUNNINGHAM, D., MILLER, C. G., XUE, Z., ZHOU, J-M., AND BOYER, P. D. (1987). Adenine nucleotide binding sites on beef heart Fl ATPase: Photoaffinity labeling of o-subunit Tyr-368 at a noncatalytic site and fi Tyr-345 at a catalytic site. Proc. Natl. Acad. Sci. USA 84: 5715-5719. WEINBERG,

A. P., AM) VOGELSTEIN,

B. (1983).

A technique

for

MAMMALIAN

11.

12.

13.

14.

15.

16. 17.

18. 19.

HEXOKINASE

1: EVOLUTIONARY

radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. B&hem. 132: 6-13. FITCH, W. M., AND MARCOLIASH, E. (1969). Construction of phylogenetic trees: A method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155: 279-284. FLAHERTY, K. M., DELUCA-FLAHERTY, C., AND MCKAY, D. B. (1990). Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346: 623-628. FROHLICH, K-U., ENTIAN, K-D., AND MECKE, D. (1985). The primary structure of the yeast hexokinase PI1 gene (HXKP) which is responsible for glucose repression. Gene 36: 105-111. FROHMAN, M. A., DUSH, M. K., AND MARTIN, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998-9002. FRY, D. C., KUBY, S. A., AND MILDVAN, A. S. (1986). ATPbinding site of adenylate kinase: Mechanistic implications of its homology with ras-encoded ~21, Fl-ATPase, and other nucleotide binding proteins. Proc. Natl. Acad. Sci. USA 83: 907-911. GEORGE, D. G., BARKER, W. C., AND HUNT, L. T. (1986). The protein identification resource (PIR). Nucleic Acids Res. 14: 11-20. GRIFFIN, L. D., MACGREGOR, G. R., MUZNY, D. M., HARTER, J., COOK, R. G., AND MCCABE, E. R. B. (1989). Synthesis and characterization of a bovine HKl cDNA probe by mixed oligonucleotide primed amplification of cDNA using high complexity primer mixtures. Biochem. Med. Metub. Biol. 41: 125131. GROSSBARD, L., AND SCHIMKE, R. T. (1966). Multiple hexokinases of rat tissues. Purification and comparison of soluble forms. J. Biol. Chem. 241: 3546-3560. GYLLENSTJZN, U. B., AND ERLICH, H. A. (1988). Generation of single-stranded DNA by the polymerase chain reaction and its application to sequencing of the HLA-DQA locus. Proc. Natl.

Acad.

Sci.

28. 29. 30.

31.

32.

33.

34.

35.

36.

USA 85: 7562-7566.

20. HANKS, S. K., QUINN, A. M., AND HUNTER, T. (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52. 21. HANNINK, M., AND DONOGHUE, D. J. (1985). Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation. Proc. Natl. Acad. Sci. USA 82: 7894-7898. 22. HARRISON, R. W. (1985). “Crystallographic Refinement of Two Isozymes of Yeast Hexokinase and the Relationship of Structure to Function.” PhD. thesis, Yale University, New Haven, CT. 23. HASHIMOTO, E., TAKIO, K., AND KREBS, E. G. (1982). Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase. J. Biol. Chem. 257: 727-733. 24. HERRERO, P., FERNANDEZ, R., AND MORENO, F. (1989). The hexokinase isozyme PI1 of Sacchnromyces cereuisiae is a protein kinase. J. Gen. Microbial. 135: 12091216. 25. HOLROYDE, M. J., AND TRAYER, I. P. (1976). Purification and properties of rat skeletal muscle hexokinase. FEBS Lett. 62: 215-217. 26. KAIMPS, M. P., AND SEPIY)N, B. M. (1986). Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p6v”. Mol. Cell. Biol. 6: 751-757. 27. KAMPS, M. P., TAYLOR, S. S., AND SEFTON, B. M. (1984).

38.

39. 40. 41.

42.

1023

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinases have homologous ATPbinding sites. Nature 310: 589-591. KATZEN, H. M., AND SCHIMKE, R. T. (1965). Multiple forms of hexokinase in the rat: Tissue distribution, age dependency, and properties. Proc. Natl. Acad. Sci. USA 54: 1218-1225. KIMURA, M. (1981). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequence. J. Mol. Euol. 16: 111-120. KOPETZKI, E., ENTIAN, K-D., AND MECKE, D. (1985). Complete nucleotide sequence of the hexokinase PI gene (HXKl) of Saccharomyces cerevisiue. Gene 39: 95-102. KUSUKAWA, N., UEMORI, T., ASADA, K., AND KATO, I. (1990). Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. Biotechniques 9: 66-72. LOWRY, 0. H., AND PASSONNEAU, J. V. (1972). “A Flexible System of Enzymatic Analysis,” pp. 121-128, Academic Press, New York. MANIATIS, T., FR~SCH, E. F., AND SAMEIROOK, J. (1982). “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. NEMAT-GORGANI, M., AND WILSON, J. E. (1986). Rat brain hexokinase: Location of the substrata nucleotide binding site in a structural domain at the C-terminus of the enzyme. Arch. Biochem. Biophys. 251: 97-103. NISHI, S., SEIKO, S., AND BELL, G. I. (1988). Human hexokinase: Sequences at the amino- and carboxy-terminal halves are homologous. Biochem. Biophys. Res. Commun. 157: 937943. SCHIRCH, D. M., AND WILSON, J. E. (1987). Rat brain hexokinase: Amino acid sequence at the substrate hexose binding site is homologous to that of yeast hexokinase. Arch. Biothem.

37.

CONSERVATION

Biophys.

254:

385-396.

SCHWAB, D. A., AND WILSON, J. E. (1989). Complete amino acid sequence of the rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase. Proc. Natl. Acad. Sci. USA 86: 2563-2567. SCHWAB, D. A., AND WILSON, J. E. (1991). Complete amino acid sequence of the type III isoxyme of rat hexokinase, deduced from cloned cDNA. Arch. Biochem. Biophys. 285: 365370. SHOHAM, M., AND STEITZ, T. A. (1980). Crystallographic studies and model building of ATP at the active site of hexokinase. J. Mol. Biol. 140: 1-14. SMITH, R. F., AND SMITH, T. F. (1990). Automatic generation of primary sequence patterns from sets of related protein sequences. Proc. Natl. Acad. Sci. USA 87: 118-122. SNYDER, M. A., BISHOP, J. M., MCGRATH, J. P., AND LEVINSON, A. D. (1985). A mutation at the ATP-binding site of pp60”-@, abolishes kinase activity, transformation, and tumorigenicity. Mol. Cell. Biol. 5: 1772-1779. STERNBERG, M. J. E., AND TAYLOR, W. R. (1984). Modelling the ATP-binding site of oncogene products, the epidermal growth factor receptor, and related proteins, FEBS Z&t. 175: 387-396.

43. TAMURA, J. K., LADINE, J. R., AND CROSS, R. L. (1988). The adenine nucleotide binding site of yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal5’-diphospho-5’-adenosine. J. Biol. Chem. 263: 7907-7912. 44. THELEN, A. P., AND WILSON, J. E. (1991). Complete amino acid sequence of the type II isozyme of rat hexokinase, de-

1024

45.

GRIFFIN

duced from a cloned cDNA: Comparison with a hexokinase from Novikoff ascites tumor. Arch. Biochem. Biophys. 286: 645-651. URETA, T. (1975). Phylogeny, ontogeny, and properties of the hexokinases from vertebrates. In “Isozymes. III. Developmental Biology” (C. L. Markert, Ed.), pp. 575-602, Academic Press, New York.

46.

URETA, T. (1982). brate hexokinases.

47.

VANDERCAMMEN, A., AND VAN SCHAY~INGEN, E. (1990). mechanism by which rat liver glucokinase is inhibited regulatory protein. Eur. J. Biochem. 191: 483-489.

48.

Vow-s, D. T., AND EASTERBY, J. S. (1979). Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors. Biochim. Biophys. Acta 566: 283295. WALKER, 3. E., SARASTE, M., RUNSWICK, M. J., AND GAY,

49.

The comparative Comp. Biochem.

isozymology Physiol. 71B:

of verte545-555.

ET

AL. N. J. (1982). Distantly related sequences in the cy- and p-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and a common nucleotide binding protein. EMBO J. 8: 945-951.

50.

WHITE, T. K., AND WILSON, J. E. (1987). Rat brain hexokinase: Location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme. Arch. Biochem. Biophys. 259: 402-411.

51.

WHITE, T. K., AND WILSON, J. E. (1989). Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: Retention of full catalytic activity in the isolated C-terminal half. Arch. Biochem. Biophys. 274: 375393.

52.

ZOLLER, M. J., NELSON, N. C., AND TAYLOR, S. S. (1981). Affinity labeling of CAMP-dependent protein kinsse with pfluorosulfonylbenzoyl adenosine. J. Bid. Chem. 266: 18371842.

The by a