Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

150

[16]

ENZYME CLONING, EXPRESSION, AND PURIFICATION

[ 16] H u m a n

3-Hydroxy-3-methylglutaryl-CoA

Lyase

B y H E N R Y M . M I Z I O R K O , C H A K R A V A R T H Y N A R A S I M H A N , and JACQUELINE R . ROBERTS

Introduction 3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyzes the cleavage of HMG-CoA to form acetyl-CoA and acetoacetate [Eq. (1)]. OH

0

HOOC-- CH2 -- C -- CH2-- C -- SCoA

)

I CH3

O

II CH3--

C--CH2--COOH

O

II + CH3-- C--SCoA

(1)

This reaction is a key step in ketogenesis and also represents the last step in the leucine catabolic pathway. Deficiencies in enzyme activity account for the human inherited metabolic disease, hydroxymethylglutaric aciduria. 1 Although enzyme preparations from mammalian liver have been reported and characterized, 2'3 the first homogeneous, high specific activity preparation from eukaryotic tissue was reported for the avian enzyme. 4 This protein, as well as a homogeneous preparation of the recombinant Pseudomonas mevalonii enzyme, 5 were used in affinity labeling studies that identified elements of the catalytic apparatus. 6 Work with the avian enzyme also suggested that an intersubunit thiol/disulfide exchange represents a potential mechanism for regulating activity of the eukaryotic enzyme. 7 Investigation of catalytic and potential regulatory mechanisms, as well as the modeling of mutations that result in human hydroxymethylglutaric aciduria, required the development of a convenient recombinant source of stable, homogeneous human enzyme. For this reason, human lyase-encod-

1 K. M. Gibson, J. Breuer, and W. L. Nyhan, Eur. J. Pediatr. 1411, 180 (1988). 2 B. K. Bachawat, W. G. Robinson, and M. J. Coon, J. Biol. Chem. 216, 727 (1955). 3 L. D. Stegink and M. J. Coon, J. Biol. Chem. 243, 5272 (1968). 4 p. R. Kramer and H. M. Miziorko, J. Biol. Chem. 2,55, 11023 (1980). 5 C. Narasimhan and H. M. Miziorko, Biochemistry 31, 11224 (1992). 6 p. W. Hruz, C. Narasimhan, and H. M. Miziorko, Biochemistry 31, 6842 (1992). 7 p. W. Hruz and H. M. Miziorko, Protein Sci. 1, 1144 (1992).

METHODS IN ENZYMOLOGY.VOL. 324

Copyright© 2000 by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/00 $30.00

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ing c D N A 8 was used to develop a bacterial expression system for the mature mitochondrial form of human H M G - C o A lyase. 9 The methodology described in this account is used for routine expression and isolation of the active recombinant mitochondrial isoform of the enzyme. Subsequently, analogous approaches were used to produce a protein containing the leader sequence that is proteolysed concomitant with import of H M G - C o A lyase into the mitochondrion; this full-length protein appears to represent a peroxisomal isoform of the enzyme. 1° Isolation of this isozyme has been reported; the protocoP 1 involves only slight modifications of the procedure outlined for the mitochondrial isoform. Assay M e t h o d s

Principle H M G - C o A cleavage may be followed spectrophotometrically by using coupled enzyme assays that measure production of either of the two reaction products, acetyl-CoA (citrate synthase-coupled assay) or acetoacetate (/3hydroxybutyrate assay). In addition, the reaction may be followed with increased sensitivity using [14C]HMG-CoA, estimating substrate cleavage by measuring conversion of the acid-stable radioactivity of the substrate to a volatile radiolabeled product. Each of these assays has been required in different experimental situations; procedures for each assay are documented in this volume 12 in [15], on P. mevalonii H M G - C o A lyase. To support the isolation of recombinant human mitochondrial H M G - C o A lyase, the citrate synthase-coupled spectrophotometric assay has been routinely used; this assay is described below.

Cim~te Synthase-Coupled Assay of HMG-CoA Lyase Activity Reagents Tris-HC1 (pH 8.2), 1.0 M MgCI2, 1.0 M 8 G. A. Mitchell,M. F. Robert, P. Hruz, S. Wang, G. Fontaine, C. Behnke, L. Mende-Mueller, K. Schappert, C. Lee, K. M. Gibson, and H. Miziorko,J. Biol. Chem. 268, 4376 (1993). 9j. R. Roberts, C. Narasimhan, P. W. Hruz, G. A. Mitchell, and H. M. Miziorko, J. Biol. Chem. 269, 17841 (1994). 10L. Ashmarina, N. Rusnak, H. M. Miziorko, and G. A. Mitchell, J. Biol. Chem. 269, 31929 (1994). 11L. Ashmarina, M. F. Robert, M. A. Elsliger, and G. A. Mitchell, Biochem. J. 315, 71 (1996). 12H. M. Miziorko and C. Narasimhan, Methods Enzymol. 324, Chap. 15, 2000 (this volume).

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ENZYME CLONING, EXPRESSION, AND PURIFICATION

[I 6]

Dithiothreitol (DTF), 100 mM NADH, 5 mM NAD, 30 mM L-Malate, 50 mM (neutralized as potassium salt) HMG-CoA, 6 mM, pH 4.5 Citrate synthase (pig heart), 215 U/mg Malate dehydrogenase (pig heart), 900 U/mg Procedure. This spectrophotometric assay represents a modification 4 of the method of Stegink and Coon, 3 in which acetyl-CoA, produced along with acetoacetate on HMG-CoA cleavage, is coupled to a citrate synthase assay. Reaction of acetyl-CoA to produce citrate requires oxaloacetate, generated in the cuvette by malate dehydrogenase-catalyzed oxidation of malate with the reduction of NAD ÷ and the resulting increase in A340n m . The rate of NAD + reduction is proportional to the amount of HMG-CoA lyase added to a limiting value of 1.5 AA/min and linear until a total absorbance increase of >0.4 has occurred. The complete reaction mixture (1.0 ml) contains 200/zmol of Tris-HC1 (pH 8.2), 10/zmol of MgC12, 1.5 txmol of N A D ÷, 0.05/zmol of NADH, 5.0/zmol of DTT, 2.5/zmol of Lmalate, malate dehydrogenase (9 U), citrate synthase (4 U), and the sample containing HMG-CoA lyase. After the mixture has been incubated at 30 ° and a stable baseline recorded, HMG-CoA (0.12/zmol) is added and the rate of increase in A340nm is measured. An extinction coefficient of 6.2 × 103 M -1 is used to quantitate product acetyl-CoA formation. This assay is used routinely for measuring HMG-CoA lyase activity at stages of enzyme isolation where N A D H oxidase contaminants do not require significant correction of the measured rate of absorbance increase. The small volumes of Escherichia coli extracts needed to measure activity of recombinant HMG-CoA lyase do not cause serious interference. This assay may be unsuitable for inhibition studies that involve acyl-CoA analogs that also significantly inhibit citrate synthase or for evaluation of alternative substrates that produce an acyl-CoA product that does not couple with citrate synthase. Units. A unit of enzymatic activity is defined as the amount of enzyme necessary to convert 1 /xmol of HMG-CoA to products acetyl-CoA and acetoacetate in 1 rain under the conditions described. Specific activity is expressed in units per milligram of protein. In the published reports from which these methods have been compiled, protein concentration has been determined by the Bradford method 13 with bovine serum albumin used as a calibration standard.

13 M. M. Bradford, A n a l Biochem. 72, 248 (1976).

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HUMAN 3-HYDROXY-3-METHYLGLUTARYL-CoALYASE

153

Design of E x p r e s s i o n Plasmid for H u m a n Mitochondrial HMG-CoA Lyase In designing an expression system, it is necessary to decide whether to insert into the expression vector the complete cDNA, encoding the transit peptide-containing precursor to the mature mitochondrial matrix protein, or a shortened sequence encoding a protein closer in sequence to the mature H M G - C o A lyase. Catalytically active precursor forms of mitochondrial matrix enzymes have been produced 14'15 by recombinant D N A methodology. However, our laboratory has directly determined the N terminus of the mature avian H M G - C o A lyase. 9 Alignment of these data with the deduced sequences of the human s and mouse 16 precursor proteins has indicated sufficient homology to allow the assignment of T28 as the N terminus of the mature human enzyme. The M26G27 sequence of the precursor protein suggests the mutation of the C T A T G G sequence in the c D N A (bases 74-79) to a C C A T G G sequence, which encodes an N c o I cleavage site that overlaps a start codon. This change is implemented by polymerase chain reaction (PCR) mutagenesis techniques. 17 The modified c D N A is thus predicted to encode a protein with a two-amino acid (Met-Gly) extension in comparison with the mature mitochondrial lyase. Because N-terminal methionines are typically cleaved from proteins expressed in E. coli, a single glycine residue extension is anticipated for the purified recombinant lyase. In view of the fact that slight heterogeneity at the extreme N terminus is apparent on comparison of deduced sequences for bacteria, TM cricket, s mouse, 16 and human enzymes, 8 this modest structural perturbation does not raise serious concerns over consequences to the structural integrity and catalytic activity of the recombinant protein. The last issue addressed in design of the expression system involves changing the G A A G C C sequence (bases 994-999) to G G A T C C by PCR mutagenesis. This produces a B a m H I site downstream from the stop codon (bases 976-978) in the cDNA.

14F. Altieri, J. R. Mattingly, Jr., F. J. Rodriguez-Berrocal,J. Youssef, A. Iriarte, T. Wu, and M. Martinez-Carrion,J. Biol. Chem. 264, 4782 (1989). 15j. Jeng and H. Weiner, Arch Biochem. Biophys. 289, 221 (1991). 16S. Wang, J. H. Nadeau, A. Duncan, M. F. Robert, G. Fontaine, K. Schappert, K. R. Johnson, E. Zietkiewicz, P. Hruz, H. Miziorko, and G. A. Mitchell, Mammalian Genome 4~ 382 (1993). 17S. N. Ho, H. D. Hunt, R. M. Horton, J. K. PuUen, and L. R. Pease, Gene 77, 51 (1989). 18D. H. Anderson and V. W. Rodwell, J. Bacteriol. 1/1, 6468 (1989).

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ENZYME CLONING, EXPRESSION, AND PURIFICATION

[161

Expression of Mitochondrial HMG-CoA Lyase Bacterial Growth Escherichia coil BL21(DE3) is used when transformation involves a pET-3d-derived expression plasmid. Strain JM105 is used for transformation involving the pTrc99A-derived plasmid. Bacteria are grown at the indicated temperatures in LB broth [10 g of Bacto tryptone (Difco, Detroit, MI), 5 g of Bacto yeast extract, and 5 g of NaCI per liter] supplemented with ampicillin (50 tzg/ml). Growth is monitored by measurement of optical density (OD) at 600 nm. After cultures reach an OD of -0.6, expression of HMG-CoA lyase is induced by addition of isopropyl-/3-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Bacteria are harvested at late log phase; induction at 22 ° typically requires overnight growth before harvesting. Cells are harvested by centrifugation (4 °) at 3000g for 45 min. Pellets are stored at - 2 0 ° prior to cell lysis with a French pressure cell. Construction and Evaluation of Human HMG-CoA L yase Expression Plasmids

Expression of the eDNA for human HMG-CoA lyase, modified as described above to generate NcoI and BamHI restriction sites, has been attempted with two different vectors. Into the NcoI/BamHI-digested T7 expression plasmid, pET-3d, is ligated the appropriately digested human lyase eDNA, generating pETHL-1. Similarly, the doubly digested human lyase eDNA is ligated into the NcoI/BamHI-digested plasmid pTrc99A (Pharmacia, Piseataway, N J) to generate the expression plasmid pTrcHL1 (Fig. 1). In these expression plasmids, the transcription of the lyase gene is under the control of the lac and trc promoters, respectively, and thus inducible by IPTG. On the basis of our earlier success in producing active recombinant bacterial HMG-CoA lyase5 using expression that relied on T7 polymerase, the expression vector pET-3d, which contains the appropriate NcoI and BamHI sites, was initially selected for insertion of the modified lyaseencoding eDNA insert (Fig. 1). The resulting expression plasmid (pETHL1) is transformed into E. coil BL21. As observed earlier in our expression of recombinant bacterial lyase, growth and expression of the transformed bacteria at 37° result in recovery of the target protein as a large fraction of total E. coil protein, but most of the expressed target is insoluble and inactive. In contrast to the substantial improvement in recovery of active, soluble lyase that we enjoyed when the bacterial lyase was expressed at lower temperatures, only modest improvement is observed when a similar strategy is applied to production of recombinant human HMG-CoA lyase

[16]

HUMAN 3-HYDROXY-3-METHYLGLUTARYL-CoA LYASE C

155

AC

ATCTATGGGCACTTTACCAAAG H

G

T

L

P

K

~TGAAA

TGTAAACTCTGAGC C C C T T G C C C A C C T G G ~ T T C C T

R

C

V

K

K

L

*

v

v

t Kb

I

I

E 0.0

It E 1.50

H

0.50

i. 00

--> HLH31

HLH32

ATCCATGGGCA~-I-I-I'ACCAAAGCGGGTGA~A M

G

T

L

P

K

•

t E

Kb

0.0

t !I

R

V

TGTAAACTCTGAGCCCCTTGCCCAC C T G G A T e C C T

K

C

I

K

L

*

t H

0.50

I

It

tl B

E 1.50

1.00

/\,

N

N

N

FIG. 1. Construction of plasmids for expression of human HMG-CoA lyase in E. coli.

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ENZYME CLONING, EXPRESSION, AND PURIFICATION

[16]

TABLE I EXPRESSION OF RECOMBINANT HUMAN H M G - C o A LYASE UNDER VARIOUS GROWTH CONDITIONSa

Expression plasmid

Temperature (growth/induction)

Activity (units/ml culture)

pETHL-1

370/37 ° 220/22 ° 22°/15 ° 22°/12 ° 15°/15 ° 370/37 ° 37o/30 ° 37o/22 °

0.2 0.7 0.5 0.6 0.6 0.8 1.2 2.3

pTrcHL-1

a Reprinted, with permission, P. W. Hruz, G. A. Mitchell, 269, 17841 (1994).

from J. R. Roberts, C. Narasimhan, and H. M. Miziorko, J. Biol. Chem.

in this expression system (Table I). There is precedent 19 suggesting that such problems may be circumvented by using a different expression vector; different expression rates and levels of target may correlate with recovery of a higher percentage of total expressed target protein as active, soluble enzyme. The expression vector pTrc99A, which conveniently contains unique and appropriately positioned NcoI and BamHI sites, affords a straightforward way to test this possibility. The same coding insert is, therefore, ligated into appropriately restricted vector and the resulting expression plasmid (pTrcHL-1; Fig. 1) is transformed into E. coli JM105. Although IPTG induction of the transformed bacteria does not result in overexpression of lyase at the exaggerated levels that are supported by the pET system, a significant amount of lyase protein is produced. Regardless of induction temperature (Table I), most of the expressed lyase is soluble and active, accounting for a considerable improvement in recovery of enzyme units in comparison with the pETHL-1 expression system. This observation suggests that the pTrcHL-1 system might be exploited for production of high specific activity human HMG-CoA lyase. Construction of a C323S mutant form of human HMG-CoA lyase has been described. 9 This protein lacks a cysteine close to the C terminus 19 Z. Zhang, G. Bai, S. Deans-Zirattu, M. F. 1484 (1992).

Browner, and E.

Y. C. Lee, J. Biol. Chem. 267,

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HUMAN3-HYDROXY-3-METHYLGLUTARYL°CoALYASE

157

of the protein; this residue has been implicated in a thiol/disulfide exchange that markedly affects in vitro activity.7 The coding sequence has also been inserted into pTrc99A to produce expression plasmid pTrcHL-C233S. Purification of H u m a n HMG-CoA Lyase Preliminary work (not shown) has led to a purification protocol that yields less than 1 mg of highly purified human HMG-CoA lyase from 1 liter of bacteria transformed with pETHL-1. The yield of high specific activity enzyme from pTrcHL-l-transformed cells (Table I) represents about a fivefold improvement over our results with pETHL-l-based expression. Thus, the protocol outlined below has been optimized for enzyme produced by expression using PTrcHL-1. HMG-CoA lyase is purified from the pellet of a pTrcHL-l-transformed E. coli JM105 culture (1 liter) harvested by low-speed centrifugation (3000g, 45 min, 4°) at late log phase after induction with IPTG at 22 °. The pellet is resuspendcd and homogenized in 50 ml of cold (0 °) lysis buffer [10 mM potassium phosphate (pH 7.8), 5 mM EDTA, 0.1 mM phcnylmethylsulfonyl fluoride (PMSF), DNase (10 ~g/ml), and RNase (10 ~g/ml)]. The resuspended cells are lysed in a French pressure cell at 15,000 psi. The crude extract is centrifuged at 100,000g for 1 hr at 4°. The high-speed supernatant is loaded onto a Q-Sepharose anion-exchange column (14 × 1.5 cm) equilibrated in 10 mM potassium phosphate, pH 7.8, containing 1 mM DTY (buffer A). At pH 7.8, most of the E. coli protein will bind to the anionexchange column while human HMG-CoA lyase is recovered in the unbound fraction. Protein in this recovered material is diluted to -0.30-0.40 mg/ml with buffer A. The diluted HMG-CoA lyase from the anionexchange eluate is brought to 40% (NH4)2SO4 saturation by slow addition of the salt with constant stirring at 4 °. After 2 hr of stirring, the 40% (NH4)2SO4 fraction is centrifuged at 12,100g for 20 min at 4°. The superuatant (containing the lyase activity) is immediately brought to 65% (NH4)eSO4 by addition of the solid salt with constant stirring at 4° for 15-17 hr. The precipitated HMG-CoA lyase is then centrifuged at 12,100g for 30 min at 4°. Note: Omission of DTT from the purification buffers used through this stage of the purification does not markedly affect yield or specific activity of wild-type enzyme, but D T r is absolutely necessary in the remaining purification steps. The pellet from the ammonium sulfate precipitation procedure is resuspended in 1.0 M (NH4)zSO4 in buffer B [buffer A supplemented with 20% (v/v) glycerol]. The resulting solution is loaded onto a phenyl-agarose column (18 × 1.0 cm) that has been equilibrated in

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ENZYME CLONING, EXPRESSION, AND PURIFICATION

[I 61

1.0 M (NH4)2SO4in buffer B. The column is washed with 10 ml of the equilibrating buffer, followed by 10 ml of 0.7 M (NH4)2SO4 in buffer B. HMG-CoA lyase is eluted from the hydrophobic resin with a reverse gradient of 0.7-0.0 M (NH4)2504in buffer B (60 ml); 1.5-ml fractions are collected. Because the human lyase elutes at the low ionic strength end of the gradient, an additional 20 ml of buffer B is washed through the column to ensure complete recovery of activity. The fractions are analyzed spectrophotometrically for activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity. Those fractions containing only HMG-CoA lyase and one high molecular mass contaminant (--60kDa subunit) are pooled for further purification. The pooled fractions are concentrated to - 5 ml in an Amicon (Danvers, MA) stirred cell, and further concentrated (<2 ml) in an Amicon minicell with a YM10 membrane. The concentrated sample is loaded onto a Superose 12 (preparative grade) column (50 x 2 cm) equilibrated in 0.1 M NaC1 in buffer B. HMG-CoA lyase is eluted at a flow rate of 0.22 ml/min, and fractions of 1.2 ml are collected. The high molecular weight contaminant (>240,000 native molecular weight) that copurifies through all the prior steps elutes first from the molecular sieve column, cleanly separating from HMG-CoA lyase, which S D S - P A G E indicates to be homogeneous at this stage. The isolated human HMG-CoA lyase is stored at - 8 0 °. Enzyme recovery and specific activity at different steps in the purification are summarized in Table II. Figure 2 shows S D S - P A G E data for enzyme at various stages in the purification. The C323S mutant lyase is purified as described for wild-type recombinant enzyme. In contrast to wild-type enzyme, specific activity of C323S lyase is not substantially affected by omission of 1 mM DTT from the

TABLE II PURIFICATION OF RECOMBINANTHUMAN HMG-CoA LYASEa Purification step

Total units

Crude extract Soluble extract Q-Sepharose 40-65% (NH4)2SO4 fractionation Phenyl-agarose Superose 12

3625 3795 3467 2355 1000 932

Total protein (mg) 851 748 212 82.5 10.5 5.85

Specific activity (units/mg)

Yield (%)

4.26 5.10 16.35 28.5

100 104 96 65

95.2 159

28 26

a Reprinted, with permission, from J. R. Roberts, C. Narasimhan, P. W. Hruz, G. A. Mitchell, and H. M. Miziorko, J. Biol. Chem. 269, 17841 (1994).

[16]

n

~

3-HYDROXY-3-METHYLGLUTARYL-CoALYASE

1 2 3 4 5 6

159

78

FIG. 2. SDS-PAGE of recombinant human HMG-CoA lyases at various stages of purification. Lane 1, molecular weight standards; lane 2, bacterial cell extract; lane 3, high-speed supernatant from centrifugation at 100,000g for 1 hr; lane 4, sample after Q-Sepharose; lane 5, 40--65% ammonium sulfate fraction; lane 6, phenyl-agarose eluate; lane 7, Superose 12purified wild-type human lyase; lane 8, Superose 12-purified C323S lyase. [Reprinted, with permission, from J. R. Roberts, C. Narasimhan, P. W. Hruz, G. A. Mitchell, and H. M. Miziorko, J. BioL Chem. 269, 17841 (1994).]

buffers used for the two final purification steps. As in the case of wild-type enzyme, homogeneous enzyme (Fig. 2) is recovered from the purification protocol. Isolated C323S lyase is stable for months when stored at - 8 0 °. Table III summarizes recovery and specific activity of C323S HMG-CoA lyase at different steps in the purification.

TABLE III PURIFICATIONOF RECOMBINANTC323S HUMANHMG-CoA LYASEa Purification step

Total units

Total protein (mg)

Specific activity (units/mg)

Yield (%)

Crude extract Soluble extract Q-Sepharose 40-65% (NH4)2SO4 fractionation Phenyl-agarose Superose 12

4258 4376 4114 2335

982 768 161 60.5

4.33 5.70 25.6 38.6

100 103 97 55

1971 1756

7.9 5.05

249 348

46 41

a Reprinted, with permission, from J. R. Roberts, C. Narasimhan, P. W. Hruz, G. A. Mitchell, and H. M. Miziorko, J. Biol. Chem. 269, 17841 (1994).

160

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ENZYME CLONING, EXPRESSION, AND PURIFICATION

Characterization of Human HMG-CoA Lyase E d m a n d e g r a d a t i o n of isolated wild-type e n z y m e indicates that the Nterminal m e t h i o n i n e has b e e n cleaved f r o m a p p r o x i m a t e l y 80% of the protein, confirming the prediction p r e s e n t e d a b o v e in the outline of expression strategy. While yield of the isolated h u m a n protein (per liter of cultured bacteria) is a b o u t threefold lower t h a n experienced with r e c o m b i n a n t bacterial lyase, 5 r e c o v e r y of the higher specific activity r e c o m b i n a n t h u m a n lyase is a d e q u a t e to s u p p o r t m o s t mechanistic, protein chemistry, and even structural investigations. Such w o r k will also be facilitated by the i m p r o v e d stability exhibited by the r e c o m b i n a n t e u k a r y o t i c protein in c o m p a r i s o n with the p r o k a r y o t i c enzyme. While H M G - C o A lyase has not b e e n isolated f r o m h u m a n tissue, a c o m p a r i s o n of the properties of the r e c o m b i n a n t wild-type and C323S e n z y m e s (Table IV) with those of the protein isolated f r o m avian liver suggests that these e n z y m e s represent appropriate models. H u m a n C323S

TABLE IV PROPERTIESOF HMG-CoA LYASESa Recombinant human Property

Wild-type

C323S

Avianb

Specific activity (units/mg) Km (HMG-CoA,/~M) Km (Mn2÷, tzM) Km (Mg2+,/xM) Stoichiometry of butynoyl-CoA modification c DTT stimulation of activity Subunit molecular mass (kDa)

159 24 0.34 233 1.0

348 45 0.37 322 1.0

350 8 10 50 0.9

10-fold 31.6a 34e 48.6

2-fotd 31.6a 34e 48.6

100-fold 31.4d 27e 49.0

Native molecular mass (kDa)

a Reprinted, with permission, from J. R. Roberts, C. Narasimhan, P. W. Hruz, G. A. Mitchell, and H. M. Miziorko, J. Biol. Chem. 269, 17841 (1994). b Properties of avian HMG-CoA lyase have been described by Kramer and Mizinrko4 and Hruz et al. 6 c Stoichiometry is calculated as mol~a~l/mOlsub~it. d Molecular mass calculated ~om deduced amino acid sequence, assuming that mature enzyme has been processed to produce an N terminus comparable to that measured for avian HMG-CoA lyase (T28). e Apparent subunit molecular mass determined on the basis of mobility on SDSPAGE.

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HUMAN3-HYDROXY-3-METHYLGLUTARYL-CoALYASE

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lyase matches avian enzyme in specific activity. Wild-type human lyase, isolated by the current protocol, shows a twofold diminution in specific activity despite precautions to maintain enzyme in a reduced state. This value exceeds any other reports for lyase preparations from mammalian tissues. 2'3 The lower specific activity of wild-type lyase cannot be attributed to copurification, along with catalytically active enzyme, of protein that is inactive due to gross perturbations in structure. On parallel treatment of both wild-type and C323S lyases with 2-butynoyl-CoA, which we previously documented to function as an affinity label, 6 both enzymes are inactivated. Moreover, comparable stoichiometries of modification (1.0 per site; Table IV) are observed for both enzymes. These values are in good agreement with the estimate reported for the avian liver enzyme. Thus, despite a diminished specific activity, wild-type HMG-CoA lyase appears to possess a full complement of intact substrate-binding sites. Activity of both wild-type and C323S lyases is markedly stimulated by divalent cations. Catalytic activity afforded by Mn 2+ is comparable to that supported by the physiological cation, Mg 2+. Wild-type and C323S human lyases exhibit Km values for Mg 2+ (Table II) that are four- to sixfold higher than reported for avian enzyme. Knl values for Mn 2+ are approximately three orders of magnitude lower. The observation that a cation activator such as Mn 2+ binds with such high affinity has proven useful in the design of studies 2° that identified amino acid H235 as a ligand for this cation. Recombinant human lyases exhibit, on SDS-PAGE, a subunit molecular mass that is in reasonable aggreement with the cDNA-deduced value of 31.6 kDa. Molecular sieve chromatography suggests that the native enzyme, is a dimer, as is the avian enzyme. 4 The validity of this assignment is also suggested by the results of protein cross-linking experiments. 9 Oxidation of C323, or modification of this residue with the bifunctional reagent o-phenylenedimaleimide, results in conversion of wild-type lyase to a species that exhibits, on SDS-PAGE, a 65-kDa dimer instead of the 32-kDa monomer observed with untreated, reduced enzyme. Similar treatment of C323S lyase does not produce a 65-kDa protein species. Acknowledgments Human HMG-CoA lyase studies performed in the authors' laboratory have been supported by NIH DK-21491. Collaborative interaction with the laboratory of Dr. Grant A. Mitchell (Hopital Ste.-Justine, University of Montreal) led to the isolation of cDNA encoding human HMG.-CoA lyase; the contributions of these colleagues are gratefully acknowledged. 20j. R. Roberts and H. M. Miziorko, Biochemistry 36, 7594 (1997).