Human hexokinase: Sequences of amino- and carboxyl-terminal halves are homologous

Vol. 157, No. 3, 1988

BIOCHEMICAL AND 81OPHYSICAL RESEARCH COMMUNICATIONS Pages 937-943

December 30, 1988

HUMAN

HEXOKINASE:

SEQUENCES HALVES

Shigeo Nishi,

OF AMINO-

ARE

AND

CARBOXYL-TERMINAL

HOMOLOGOUS

Susumu Seino and Graeme I. Bell

Howard Hughes Medical

Institute,

Departments of B i o c h e m i s t r y and M o l e c u l a r Biology and M e d i c i n e The U n i v e r s i t y of Chicago 5841 S. M a r y l a n d Ave., Chicago,

IL

Box 391

60637

Received September 27, 1988

SUMMARY cDNA clones encoding human hexokinase have been isolated from an adult kidney library. A n a l y s i s of this 917 amino acid p r o t e i n (Mr = 102,519) indicates that the sequences of the NH 2and COOH-terminal halves, corresponding to the r e g u l a t o r y and catalytic domains, respectively, are homologous; and that eukaryotic h e x o k i n a s e s evolved by d u p l i c a t i o n of a gene e n c o d i n g a p r o t e i n of "450 amino acids. The C O O H - t e r m i n a l half of the p r o t e i n created by this gene d u P l i c a t i o n retained the glucose b i n d i n g site and glucose p h o s p h o r y l a t i n g activity while the substrate binding sites of the N H 2 - t e r m i n a l half evolved into a new a l l o s t e r i c effector site. ©1988AcademicPress,lnc.

The ~ontrol t ere

enzymatic point

only and

in glycolysis.

of

glucose

In humans,

I,

II,

III

and

is

a

principal

rats and other mammals,

is a family of glucose p h o s p h o r y l a t i n g

hexokinase have

phosphorylation

glucokinase

enzymes,

(1-3).

The

designated hexokinases

a m o l e c u l a r mass of ~i00,000 daltons whereas g l u c o k i n a s e ~50,000 tissue

regulated

protein

disorder.

different

in in

diabetes its

or As

phosphorylating

In

an

mellitus,

abnormal

perhaps a

addition,

mechanisms.

metabolism

having

development

Each enzyme has unique kinetic properties

distribution. by

perturbed involved

daltons.

is

first enzymes,

to

we

As

we

are

determine

sequence

influence step

the

in

glucose

the

each

is

metabolism

is

examining if

the

could

of

key

contribute

the

isolated

enzymes

synthesis

pathophysiology

examining

have

activity

role

and

to of

of

of

a

the this

glucose

characterized

normal adult kidney cDNA clones encoding human hexokinase.

937

0006-291 X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

General Methods. Standard procedures were carried out as d e s c r i b e d in M a n i a t i s et al. (4). D N A s e q u e n c i n g w a s d o n e b y the dideoxynucleotide-chain-termination procedure (5) after subcloning appropriate DNA fragments into M l 3 m p l 8 or M l 3 m p l 9 . T h e s e q u e n c e s of b o t h s t r a n d s a n d a c r o s s all r e s t r i c t i o n sites were determined.

c D N A Cloning. O n e m i l l i o n p h a g e f r o m an a d u l t h u m a n k i d n e y c D N A library having inserts >2 kbp and consisting of "250,000 different clones (6) w e r e screened with a 32p-labeled EcoRI fragment encoding nucleotides 1-2114 [the n u m b e r i n g is from S c h w a b and W i l s o n (7)] of t h e c a t a l y t i c d o m a i n of a rat b r a i n hexokinase I cDNA (prHKl-l, unpublished). This probe was i s o l a t e d f r o m a rat b r a i n c D N A l i b r a r y (Clontech, RLI002, Palo Alto, CA) u s i n g s p e c i f i c o l i g o n u c l e o t i d e p r o b e s b a s e d u p o n the c D N A s e q u e n c e r e p o r t e d b y S c h w a b and W i l s o n (7). P r i o r to the publication of t h e p a r t i a l r a t h e x o k i n a s e I c D N A sequence, we were attempting to isolate hexokinase cDNA clones using oligonucleotide probes based upon the fragmentary amino acid sequence that was available (8) b u t w i t h o u t success. T h e rat b r a i n c D N A p r o b e w a s h y b r i d i z e d to the h u m a n c D N A l i b r a r y u s i n g low stringency conditions: 37°C; 25% formamide, 5 X SSC, 2 X D e n h a r d t ' s solution, 2 0 m M s o d i u m p h o s p h a t e buffer, p H 6.5, 0.1% N a D o d S O 4 , i00 # g / m l of s o n i c a t e d and d e n a t u r e d s a l m o n t e s t e s DNA, 10% d e x t r a n s u l f a t e a n d 1 X 106 c p m / m l of probe, t h e f i l t e r s w e r e w a s h e d in 2 X SSC and 0.1% N a D o d S O 4 at r o o m t e m p e r a t u r e a n d t h e n for o n e h o u r at 4 0 ° C b e f o r e a u t o r a d i o g r a p h y .

RESULTS Sequence in

the

of H u m a n adult

hexokinase of

these

complete 358

human

i)

kidney

I c D N A probe. IhHEX-12

and

sequence

nucleotides

(Fig.

Hexokinase.

of of

contained

nucleotides

encoding

well

bp

as

81

respectively. 93.4% (7), 85.3%

similarity we believe nucleotide

library

Eighteen -15

a

was

765

with

large

amino

bp

of

largest

composite

protein

have

is h u m a n between

phage

the

further

inserts. well

3,598

as

bp

frame

(M r

89.2%

the

5'

2,751 as

sequence, and

I sequence I

(there

corresponding

is

coding

Figure I. Composite nucleotide sequence of human kidney hexokinase cDNA and predicted amino acid sequence of the protein. The number of the nucleotide at the end of each line is indicated. The insert in IhHEX-15 includes nucleotides 38-3598. The 5'-EcoRI fragment of IhHEX-12, nucleotides 1-358, was also sequenced.

938

and

sequence of

identity

hexokinase hexokinase

rat

The

= 102,519)

3'-untranslated

rat b r a i n

identity

as

open-reading

467-917

this protein

sequence

with

characterized

5'-and

the partial

1 X 106

hybridized

the

acid

acids

of the

determined

The

single

a 917

were

contained

lhHEX-12.

amino

that

cDNA

IhHEX-15

and As

Seventy-nine

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS i Met Ile Ala A1a Gln Leu L e u Ala Tyr ATG A?C CCC GCG CAG CTC CTG GCC TAT

108

10 20 30 Tyr Phe ThE GIu Leu Lye Asp Asp Gln Val Lys Lys Ile Asp bys Tyr Leu Tyr Ale Met Arg Leu SeE Asp G1u ThE Leu Ile Asp Ile TAC TTC ACG GAG CTG AAG GAT GAC CAG GTC AAA AAG ATT GAC AAG TAT CTG TAT GCC ATG CGG CTC TCC GAT GAA ACT CTC ATA GAT ATC

198

40 50 00 Met ThE Arg Phe Azg Lys Glu Met Lys Asn Gly Leu SeE Arg ASp Phe ASh Pro ThE Ala ThE Val Lys Met LeU Pro ThE Phe Val Arg ATG ACT CGC TTC AGG AAG GAG ATG AAG AAT GGC CTC TCC CGG GAT TTT AAT CCA ACA GCC ACA GTC AAG ATG TTG CCA ACA TTC GTA AGG

208

70 80 90 Ser Ile Pro ASp Gly SeE Glu LyS Gly Asp Phe Ile Ala Leu ASp Leu Gly Gly Ser SeE Phe Arg Ile Leu Arg Val Gln val ASh His TCC ATT CCT GAT GGC TCT GAA AAG GGA GAT TTC ATT GCC CTG GAT CTT GGT GGG TCT TCC TTT CGA ATT CTG CGG GTG CAA GTG AAT CAT

378

IOO Ii0 120 Glu Lys Ash Gln Asn Val His Met G%u SeE Glu Val Tyr Asp ThE Pro GIu ASh Ile Val His Gly SeE Gly SeE Gln Leu Phe ASp His GAG AAA AAC CAG AAT GTT CAC ATG GHG TCC GAG GTT TAT GAC ACC CCA GAG AAC ATC GTG CAC GGC AGT GGA AGC CAG CTT TTT GAT CAT

468

130 148 150 Val Ala GIU Cys Leu G1y Asp Phe Met GIu Lys A~g Lys Ile Lys Asp LyS Lys Leu Pro Val Gly Phe Thr Phe SeE Phe P~o Cys Gln GTT GCT GAG TGC CTG GGA GAT TTC ATG GAG AAA AGG AAG ATC AAG GAC AAG HAG TTA CCT GTG GGA TTC ACG TTT TCT TTT CCT TGC CAA

558

160 170 180 Gln SeE Lys Ile Asp Glu Ala Ile Leu Ile ThE T r p ThE Lys Arg Phe 5ys Ala SeE Gly Val GI0 Gly Ala ASp Val Val Lys Leu Leu CAA TCC AAH ATA GAT GAG GCC ATC CTG ATC HCC TGG ACA AAG CGA TTT AHA GCG AGC GGA GTG GAH GGA GCA GAT GTG GTC AAA CTG CTT

648

190 200 210 ASh Lys Ala 11e Lye Lys Arg GIy ASp Tyr ASp Ala ASh lle Val Ala Val Val ASh Asp Thr val Gly ThE Met Met ThE Cys Gly Tyr AAC AAA GCC ATC AAA AAG CGH GGG GAC TAT GAT GCC AAC ATC GTA GCT GTG GTG AAT GAC HCA GTG GGC ACC ATG ATG ACC TGT GGC TAT

739

220 230 240 Asp Asp Gin His Cys GIu Val Gly Leu Ile Ile GIy Thr Gly Thr ASh Ala CyS Tyr Met GIu GIu Leu Arg His Ile Asp Leu Val Glu GAC GAC CAG CAC TGT GAA GTC GGC CTG ATC ATC GGC ACT GGC ACC HAT GCT TGC TAC ATG GAG GAA CTG AGG CAC ATT GAT CTG GTG GAA

828

250 260 270 Gly Asp GIu Gly Arg Met Cys Ile ASh ThE Glu Trp Gly Ala Phe Gly Asp ASp Gly SeE Leu GIu Asp Ile AEg Th( Glu Phe Asp AE 0 GGA GAC GAG GGG AGG ATG TGT ATC HAT ACA G A A TGG GGA GCC TTT GGA GAC GAT GGA TCA TTA GAA GAC ATC CGG ACA GAG TTT GAC AGG

918

280 290 300 GIu Ile Asp Arg Gly SeE Leu ASh Pro Gly Lye G l n Leu Phe Glu Lys Met Val Ser Gly Met Tyr Leu Gly GIu Leu Val AE 0 Leu Ile GAG ATA GAC CGG GGA TCC CTC AAC CCT GGA AAA CAG CTG TTT GAG AAG ATG GTC AGT GGC ATG TAC TTG GGA GAG CTG GTT CGA CTG ATC

1008

310 320 330 Leu vaZ Lys Met Ala LFS Glu Gly Leu Heu Phe Glu Gly Arg Ile ThE PEO Glu Leu Leu ThE Arg Gly Dys Phe Asn ThE SeE Asp Val CTA GTC AAG ATG GCC AAG GAG GGC CTC TTA TTT GAA GGG CGG ATC ACC CCG GAG CTG CTC ACC CGA GGG AAG T T T AAC HCC AGT GAT GTG

1098

340 390 360 SeE Ala Ile GIu Lys ASh Lys GIu Gly Leu His Ash Ala Lye Glu Ile Leu ThE Arg Leu Gly Val GIu Pro SeE ASp Asp A s p Cys Val TCA GCC ATC GAA AAG AAT AAG GAA GGC CTC CAC AAT GCC AAA GAA ATC CTG ACC CGC CTG GGA GTG GAG CCG TCC GAT GAT GAC TGT GTC

1188

•CGCCGGAGGAC•ACGGCTCGCCAGGG•TGCGGAGGACCGA••GTCC••ACGC•TG•CGC••CGcGACCC•GA•CGCCAGC

g~ Val

390

990

Gln His Val Cys ThE Ile Val SeE Phe Arg SeE Ala ASh Leu Val Ala Ala Thr Leu G1y Ale Ile Leu Asn keg Leu Ar 9 Asp TCA GTC CAG CAC GTT TGC ACC ATT GTC TCA TTT CGC TCA GCC AAC TTG GTG GCT GCC ACA CTG GGC GCC ATC TTG AAC CGC CTG CGT GAT

1278

400 410 420 Asn LyS Gly ThE P~o Arg Leu Arg Thr Thr Val Gly Val Asp Gly SeE Leu Tyr LyS ThE His Pro Gln TyE Ser Arg Arg Phe His Hys AAC AAG GGC ACA CCC AGG CTG CGG ACC ACG GTT GGT GTC GAC GGA TCT CTT TAC AAG ACG CAC CCA CAG TAT TCC CGG CGT TTC CAC HAG

1368

430 440 450 ThE LeO Arg Ar 9 Leu Val Pro ASp SeE ASp Val Arg Phe Leu Leu SeE Glu Ser Gly Ser Gly Lys Gly Ala Ala Met Val ThE Ala Val ACT CTA AGG CGC TTG GTG CCA GAC TCC GAT GTG CGC TTC CTC CTC TCG GAG AGT GGC AGC GGC AAG GGG GCT GCC ATG GTG ACG GCG GTG

1458

460 470 480 Ala Tyr Arg Leu Ala Glu Gln His Arg Gln Ile GIu GIu ThE Leu Ala 81s Phe His Leo Thr Lys Asp Met Leu Leu Glu Val Lys Lys GCC TAC CGC TTG GCC GAG CAG CAC CGG CAG ATA GAG GAG ACC CTG GCT CAT TTC CAC CTC ACC AAA GAC ATG CTG CTG GAG GTG AAG AAG

1548

490 500 530 Arg Met Arg Ale GIU Met Glu Leu Gly Leu Arg Lys Gin ThE Hie Hsn ASh Ala Val Val Lys Set Leu P~o Ser Phe Val AEg Arg ThE AGG ATG CGG GCC GAG ATG GAG CTG GGG CTG AGG AAG CAG ACG CAC AAC AAT GCC GTG GTT AAG ATG CTG CCC TCC TTC GTC CGG AGA ACT

1638

520 530 540 Pro ASp Gly Thr GIU ASh Gly ASp Phe Leo Ala Leo Asp Leu Gly Gly ThE ASh Phe Arg Val Heu Leo Val Lys Ile Arg SeE Gly bys CCC GAC GGG ACC GAG AAT GGT GAC TTC TTG CCC CTG GAT CTT GGA GGA ACC AAT TTC CGT GTG CTG CTG GTG AAA ATC CGT AGT GGG AAA

1728

SSO 560 570 Hys Arg ThE Val GlU Met His ASh Lys Ile Tyr Ala Ile P~O Ile Glu Iie Met Gln Gly ThE Gly GIu GIU Leu Phe ASp His Ile Val AAG AGA ACG GTG GAA ATG CAC AAC AAG ATC TAC GCC ATT CCT ATT GAA ATC ATG CAG GGC ACT GGG GAA GAG CTG TTT GAT CAC ATT GTC

1819

500 590 600 Ser Cys Ile Ser Asp Phe Leu A S p Tyr Met GIy Ile Lys Gly Pro Ar 0 Met Pro Leu Gly Phe ThE Phe Ser Phe Pro Cys Gln Gln Thr TCC TGC ATC TCT GAC TTC TTG GAC TAC ATG GGG ATC AAA GGC CCC AGG ATG CCT CTG GGC TTC ACG TTC TCA TTT CCC TGC CAG CAG ACG

1908

610 630 630 SeE Leu Asp Ala Gly lle Leu Ile ThE Trp Thr Lys Gly Phe Lys Ala Thr Asp Cys Val Gly Sls Asp Val Val Thr Leu Leu Arg Asp AGT CTG GAC GCG GGA ATC TTG ATC ACG TGG ACA AAG GGT TTT AAG GCA ACA GAC TGC GTG GCC CAC GAT GTA GTC ACC T T A CTA AGG GAT

1998

640 650 660 Ala Ile Lys Arg Ar 0 Glu Glu Phe Asp Leu ASp Val Val Ala Val Val ASh Asp ThE Val Gly ThE Met Met Thr Cys Ala Tyr Glu G1u GCG ATA AAA AGG AGA GAG GAA TTT GAC CTG GAC GTG GTG GCT GTG GTC AAC GAC ACA GTG GGC ACC ATG ATG ACC TGT GCT TAT GAG GAG

2088

680 690 Th~ Cys Glu val Gly Leu Ile Val Gly Thr Gly SeE ASh Ala Cys Tyr Met Glu GIU Met Lys ASh Val Glu Met Val GIu Gly Asp CCC ACC TGT GAG GTT GGA CTC ATT GTT GGG ACC GGC AGC AAT GCC TGC TAC ATG GAG GAG ATG AAG AAC GTG GAG ATG GTG GAG GGG GAC

2178

700 710 720 Gln Gly Gln Met Cys Ile ASh Met GIu Trp Gly Ala Phe GIy Asp ASh Gly Cys Leu Asp Asp Ile Arg Thr His Tyr A s p Arg Leu Val CAG GGG CAG ATG TGC ATC AAC ATG GAG TGG GGG GCC TTT GGG GAC AAC GGG TGT CTG GAT GAT ATC AGG ACA CAC TAC GAC AGA CTG GTG

2288

670

Fro

~sn30

740 750 GIu Tyr 8er Leu ASh Ala Gly Lys Gln Arg Tyr Glu Lys Met Ile SeE Gly Met Tyr Leu Gly GIu 11e Val Arg Ash Ile Leu Ile AAC GAA TAT TCC CTA AAT GCT GGG AAA CAA AGG TAT GAG AAG ATG ATC AGT GGT ATG TAC CTG GGT GAA ATC GTC CGC AAC ATC TTA ATC

2358

760 770 780 ASp Phe Thr bys Lys Gly Phe Leu Phe Arg GIy Gln Ile SeE Glu ThE Met Lys ThE Arg Gly i l e Phe Glu Thr LyS Phe Leu SeE Gin GAC TTC ACC AAG AAG GGA TTC CTC TTC CGA GGG CAG ATC TCT GAG ACG ATG AAG ACC CGG GGC ATC TTT GAG ACC AAG TTT CTC TCT CAG

2448

790 800 810 ile Glu Ser ASp Arg Leu Ala Leu Leu Gln Val Arg Ala Ile Leo Gln Gln Leu Gly Leu Asn SeE ThE Cys ASp Asp Ser Ile Leu Val ATC GAG AGT GAC CGA TTA GCA CTG CTC CAG GTC CGG GCT ATC CTC CAG CAG CTA GGT CTG AAT AGC ACC TGC GAT GAC AGT ATC CTC GTC

2538

820 830 840 Lys ThE Val Cys Gly Val Val Se~ Arg Arg Ala Ala Gln Leu Cys Gly A1a Gly Met Ala Ala Val Val Asp Lys Ile Arg Glu ASh Arg AAG ACA GTG TGC GGG GTG GTG TCC AGG AGG GCC GCA CAG CTG TGT GGC GCA GGC ATG GCT GCG GTT GTG GAT HAG ATC CGC GAG AAC AGA

2628

850 860 870 GIy Leu Asp Arg Leu ASh Val Th~ Val G1y Val Asp Gly Thr Heu Tyr LyS Leo His P~o His Phe Ser Arg lle Met His Gln Thr Val GGA CTG GAC CGT CTG AAT GTG ACT GTG GGA GTG GAC GGG ACA CTC TAC AAG CTT CAT CCA CAC TTC TCC AGA ATC ATG CAC CAG ACG GTG

2718

880 890 900 Lys GIu Leu Ser Pro Lys Cys Ash Val Ser Phe Leu Leu SeE GIu A s p Gly SeE Gly Lys G1y Ala Ala Leu Ile ThE Ala Val Gly Val HAG GAA CTG TCA CCA AAA TGT AAC GTG TCC TTC CTC CTG TCT GAG GAT GGC AGC GGC AAG GGG GCC GCC CTC ATC ACG GCC GTG GGC GTG

2808

910 917 Arg Leu Arg ThE Glu Ala SeE SeE OC CGG TTA CGC ACA GAG GCA AGC AGC TAA GA~T~GGGAT~CCAG~CTA~T~CTCT~A~cA~TTCTCT~TT~AAG~GG~GA~C~CTA~CTc~AG~GAGT~G~TGG

2919

GAGACGCTGGCGCCAOGC-CCTGCCGGCGCGGGGAGG~AAGC~.~AATCCAACT~ATGG~ATATATTGTAGGGTACAGAATAGAG CGTGTGCTGTTGATAATATC~TCACC CGGATCCCr C 3039 CTCA CTTG CCCTG CCACTTTG CATGGTTTGATTTTG AC CTG GTCCCCCACG TGTGAAGTGTAGTG GCATC CATTTCT ~

TA~ CA~ATC

CAACAGAGTTA~TAT~

G GAAAAT CACAC CACCTGAC AGGC CTTC TGGC~CTCC AAAC~CCCATCCTTGGGGTTCCCCCTC CCTGTGTGAAATGTATTATCA C C AGCAGA CA C T G C C G G ~ C T C C C ~ C TGCCTGAAGGCGAGTGTGGGCATAGCATTAGCTG

CT TCCT CCCCT CCTG GCAC C C A C T G T G C - C C T G G C A T C G C A T C G T G ~ G T G T C ~

CC A C ~

CTAGC CGCGTGTGAC AGT CTTGCATTCTGTTTGTCT C G T G G G G G G A G G T G G A C A G T C C T G C G G A A A T G T G T C T T G T C T T C C A T T T G G A T A A A ~ A C C ~ C CTGGAATTTC CCAC CGCTTTG TGAGC CGTG TCGTATGACCT AGTAAACTTTGTAC CAATTC A ~ A A A A A A ~ A A

939

GCTG GAGAT 3159 ~GGGGCAC

3279

GTGTGTC C G ~ G A A C CAG TC 3398 C~C~AC~T~

C ATC A 3519 3588

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i MIAAQLLAYYFTELKDDQVKKIDKYLYAMRLSDETLIDIMTRFRKEMKNGLSRDFNPTATVKMLPTFVRSIP

I* I *I * *I I * l l l * * ** ** I * *****I*** * 464 - AEQHRQIEETLAHFHLTKDMLLEVKKRMRAEMELGLRKQTHNNAVVKMLPSFVRRTP 73 DGSEKGDFIALDLGGSSFRILRVQVNHEKNQNVHMESEVYDTPENIVHGSGSQLFDHVAECLGDFMEKRKIK •*I* ***I******I **I* * I * * * I* * *I *I* ****I *I **II ** 521

DGTENGDFLALDLGGTNFRVLLVKIRSGKKRTVEMHNKIYAIPIEIMQGTGEELFDHIVSCISDFLDYMGIK

145 D K K L P V G F T F S F P C Q Q S K I D E A I L I T W T K R F K A S G V E G A D V V K L L N K A I K K R G D Y D A N I V A V V N D T V G T M M T

II*I**********I I* ******* * * * I * *** ** ***I* II* I************* 593 GPRMPLGFTFSFPCQQTSLDAGILITWTKGFKATDCVGHDVVTLLRDAIKRREEFDLDVVAVVNDTVGTMMT 217 CGYDDQHCEVGLIIGTGTNACYMEELRHIDLVEGDEGRMCINTEWGAFGDDGSLEDIRTEFDREIDRGSLNP • *II ******I***I*******I[ III**** * **** ******* * *I**** I** I *** 665

CAYEEPTCEVGLIVGTGSNACYMEEMKNVEMVEGDQGQMCINMEWGAFGDNGCLDDIRTHYDRLVNEYSLNA

289

GKQLFEKMVSGMYLGELVRLILVKMAKEGLLFEGRITPELLTRGKFNTSDVSAIEKNKEGLHNAKEILTRLG

•** I***I*******I** **I * * * * * *I I *** * * I* ** I * I I ** ** 737 GKQRYEKMISGMYLGEIVRNILIDFTKKGFLFRGQISETMKTRGIFETKFLSQIESDRLALLQVRAILQQLG 361 VEPSDDDCVSVQHVCTIVSFRSANLVAATLGAILNRLRDNKGTPRLRTTVGVDGSLYKTHPQYSRRFHKTLR I I ** I * ** I** * *I* * I *II If*l'i* ** ******I*** ** I** * *II 809

LNSTCDDSILVKTVCGVVSRRAAQLCGAGMAAVVDKIRENRGLDRLNVTVGVDGTLYKLHPHFSRIMHQTVK

433

RLVPDSDVRFLLSESGSGKGAAMVTAVAYRL - 463 • * * ***** *******II*** ** ELSPKCNVSFLLSEDGSGKGAALITAVGVRLRTEASS

881

- 917

Figure 2. Comparison of the sequences of the NH 2- and COOHterminal halves of human kidney hexokinase. Amino acids are indicated by their single-letter abbreviations. The upper sequence is the NH2-terminal 463 amino acids and the lower sequence is the COOH-terminal 454 residues. Asterisks denote identical amino acids and vertical lines indicate chemically similar residues. The number of the amino acid at the beginning of each line is noted.

regions

of

the

NH2-terminus

cDNA

of

sequences).

this

direct

The

protein

is

also

of

rat

determined

by

Ile-(Ala,

Gln)-Ala-Leu-Leu-Ala-Tyr]

indicates

that

Sequences

of

It a

has

been

process

the

NH 2

readily

N H 2-

gene

and

homologous

slightly

more

to

confirms

identity

domain

respectively)

than

of with

of

similar

the

to

that

I

[Met-

hexokinase

this

halves

identity

and

also

of

is

between human

yeast

A

of

regulatory

940

B

domain

(30.8%

N H 2domain

There

and

(33.9%

(the

the

(10-12). B

and and

be

67.9%

enzyme Both

A

of

can

catalytic

hexokinases

hexokinase

respectively).

this

by

human

identity,

60.1%).

and

the

sequences

hexokinase

51.9%

COOH-terminal

hexokinases

evolved

of

The

human

is

Homologous.

have

Analysis

regions

the

are

hexokinases

There two

the

Hexokinase

suggestion.

identity

domain

brain

(1-3,7). this

2).

these

yeast

of

mammalian

duplication

nucleotide

regulatory

catalytic

that

between

very

(9);

COOH-Halves

(Fig.

sequence

NH2-terminus.

COOH-terminal

aligned

corresponding

are

and

sequence -

is t h e

suggested

similarity,

terminal

Met-i

of

hexokinase

sequencing

predicted

and

is the

35.1%, 29.0%,

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION Mammalian of

~I00,000

hexokinases

daltons.

glucose,

they

glucose-6-PO 4 .

are

subject

not

to

allosteric

By

glucose-phosphorylating

do

of a single p o l y p e p t i d e

contrast,

enzyme,

allosteric

observations

led

other and

by

or

duplication.

yeast

investigators

hexokinase

Subsequently,

ancestral

fusion

function.

The

the

the

yeast

glucose-6-PO 4 . to

postulate

m a m m a l i a n h e x o k i n a s e s evolved from an ancestral glucokinase

by

mammalian

chains of -50,000 daltons

inhibition

several

inhibition the

glucokinase,

are single p o l y p e p t i d e

show

chain

In addition to binding and p h o s p h o r y l a t i n g

product,

hexokinases

consist

by

a

and

These

that

the

enzyme similar to

process

involving

gene

one of the two catalytic sites in the

protein

evolved

studies

of

to

Wilson

acquire

and

his

a

regulatory

colleagues

have

c o n f i r m e d many of the features of this model

for the structure of

mammalian

have

hexokinase

NH2-terminal domain

and

half

(3,7-9,

of

contains

rat

the

brain

and

of

the

enzyme

ATP

(13,14).

terminal

-i0 kDa,

binding

In

They

hexokinase

allosteric

and that the COOH-terminal portion

13-15).

site

the

addition,

they

or -80 amino acids,

of brain

is

for

hexokinase

to the

binding have

the

that

the

regulatory

glucose-6-PO 4

half of the molecule

containing

shown

(15)

is the catalytic sites

shown

functions

for

that

glucose the

NH 2-

in the reversible

outer m i t o c h o n d r i a l

membrane

(8). The

human

indicates acids

hexokinase

that

was

a

halves

suggests

that

interaction from

this

657,

708

one

of

binding I

and

residues

and 742)

two

yeast

(Ser 447)

155,

the

are

260

and

GSGKGA

residues

-450

amino

and

COOH-

in

which

Figure

involved

(7)

in

the

are also evident in

binding in

The

(residues

the

of

both

the (603,

sequence 448-453

in both domains, precede

2

membrane.

and catalytic

of human hexokinase. site,

of

conserved 294)

and

NH 2-

implicated

(7)

conserved and present acidic

those

model

mitochondrial

of h e x o k i n a s e

residues

209,

domains

ATP-binding

is also

site

four

the

presented

likely

features

hexokinase

(residues

the

are with

this

protein of

hexokinase

1-15

functional

a

alignment

hexokinase

yeast

putative

896-901)

human

confirms

encoding

The

alignment;

by

regulatory

the

of

of

other

glucose

region

duplicated.

terminal

Several

gene

sequence

of and

however,

putative

ATP-

in the catalytic domain of human and rat h e x o k i n a s e hexokinases

in the

A

and

B has

regulatory domain 941

been

replaced

by

of human hexokinase.

a

serine Schwab

Vol. 157, No. 3, 1988

and Wilson

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(7) have

suggested

that this pair

might serve to orient the phosphate bound the

glucose.

Could

regulatory

substitutions

the

domain

side-chain

serine to

one

the

It

is

perhaps

phosphofructokinase in

the

and/or

duplication half

of

and

regulatory

of

and

new

of

into

It

seems may

sites

be

(phosphofructokinase)

allosteric

effector

molecules

are

a

this

to the

and

enzymes

allosteric of

gene

NH2-terminal domain,

(16)

the

evolved

process

efficient

enzymes

key

process

regulatory

monomeric

have

altered

which

whereas

an

the

hexokinase

subject

that

in both

of

of

acid

of this domain?

by

the

region

amino

both

phosphofructokinase

tetrameric substrates

and

evolved However,

divergence

effector

that

eukaryotes,

developed

half

the

evolution

glycolysis

divergence.

function.

duplication

higher

inhibition,

hexokinase

COOH-terminal

evolving

of

regulation

activation

interesting

in this

of

substrate binding and regulatory properties

residues

of ATP towards the

replacement

represent

contributing

of acidic

of

a

gene

mechanism

for

(hexokinase)

and

especially

structures

when similar

the to

for the enzyme.

ACKNOWLEDGEMENTS The assistance of Ms. Julie Dicig in the preparation of this manuscript is greatfully appreciated. This research was supported by the Howard Hughes Medical Institute.

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0.

Purich, D.L., Fromm, H.J. & Rudolph, F.B. (1973) Adv. in Enzymol. 35, 249-326 Ureta, T. (1982) Comp. Biochem. Physiol. 71B, 549-555 Wilson, J.E. (1980) Current Topics in Cellular Regulation 16, 1-44 Maniatis, T., Fritsch, E.F. & Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY Sanger, F., Coulson, A.R., Barrell, B.G., Smith, A.J.H. & Roe, B.A. (1980) J. Mol. Biol. 143, 161-178 Bell, G.I., Fong, N.M., Stempien, M.M., Wormsted, M.A., Caput, D., Ku, L., Urdea, M.S., Rall, L.B. & SanchezPescador, R. (1986) Nucleic Acids Res. 14, 8427-8446 Schwab, D.A. & Wilson, J.E. (1988) J. Biol. Chem. 263, 3220-3224 Schirch, D.M. & Wilson, J.E. (1987) Arch. Biochem. Biophys. 257, 1-12 Polakis, P.G. & Wilson, J.E. (1985) Arch. Biochem. Biophys. 236, 328-337 Frohlich, K., Entian, K. & Mecke, D. (1985) Gene 36, 105iii

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Kopetzki, E., Entian, K. & Mecke, D. (1985) Gene 39, 95102 Stachelek, C., Stachelek, J., Swan, J., Botstein, D. & Konigsberg, W. (1986) Nucleic Acids Res. 14, 945-963 Nemat-Gorgani, M. & Wilson, J.E. (1986) Arch. Biochem. Biophys. 251, 97-103 Schirch, D.M. & Wilson, J.E. (1987) Arch. Biochem. Biophys. 254, 385-396 White, T.K. & Wilson, J.E. (1987) Arch. Biochem. Biophys. 259, 402-411 Poorman, R.A., Randolph, A., Kemp, R.G. & Heinrikson, R.L. (1984) Nature 309, 467-469

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