Tài liệu Báo cáo khoa học: Use of hydrostatic pressure to produce ‘native’ monomers of yeast enolase

Use of hydrostatic pressure to produce ‘native’ monomers of yeast

enolase

M. Judith Kornblatt1

, Reinhard Lange2,* and Claude Balny2,*

1

Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada; 2

INSERM Unite 128, IFR 122,

Montpellier, France

The effects of hydrostatic pressure on yeast enolase have

been studied in the presence of 1 mM Mn2+. When com￾pared with apo-enolase, and Mg-enolase, the Mn-enzyme

differs from the others in three ways. Exposure to hydro￾static pressure does not inactivate the enzyme. If the

experiments are performed in the presence of 1 mM Mg2+,

or with apo-enzyme, the enzyme is inactivated [Kornblatt,

M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775–

780]. The UV spectra of the high pressure forms of the

Mg2+- and apo-forms of enolase are identical and distinct

from the spectrum of the form obtained in the presence of

1 mM Mn2+; this suggests that Mn2+ remains bound to the

high pressure form of enolase. With Mn-enolase, the various

spectral changes do not occur in the same pressure range,

indicating that multiple processes are occurring. Pressure

experiments were performed as a function of [Mn2+] and

[protein]. One of the changes in the UV spectra shows a

dependence on protein concentration, indicating that eno￾lase is dissociating into monomers. The small changes in the

UV spectrum and the retention of activity lead to a model

in which enolase, in the presence of high concentrations of

Mn2+, dissociates into native monomers; upon release of

pressure, the enzyme is fully active. Although further spectral

changes occur at higher pressures, there is no inactivation as

long asMn2+ remains bound.We propose that the relatively

small and polar nature of the subunit interface of yeast

enolase, including the presence of several salt bridges, is

responsible for the ability of hydrostatic pressure to disso￾ciate this enzyme into monomers with a native-like structure.

Keywords: dissociation; enolase; hydrostatic pressure; native

monomers.

Many enzymes normally exist as oligomeric proteins. In

some cases, the enzyme is a regulatory enzyme; allosteric

kinetics require multiple subunits. In other cases, the active

site is at the interface of the subunits, with two subunits each

contributing residues. In many cases, however, it is not

obvious what role is played by the oligomeric structure.

Attempts to study the relationship between oligomeric state

and the structure and function of the protein usually involve

dissociating the protein into its subunits and then compar￾ing the properties of the monomeric and oligomeric forms.

Often, the resulting monomers are catalytically inactive.

Because tertiary and quaternary structure are maintained by

similar forces, agents, such as temperature and chemical

denaturants, that disrupt quaternary structure may also

disrupt tertiary structure. Thus, when faced with inactive

monomers of an active oligomeric protein, it is difficult to

know if the conformation of the monomer has been altered

or if the quaternary structure is, in some way, necessary for

activity.

Hydrostatic pressure is a useful tool for studying protein

structure and function. If an equilibrium system, A Ð B, is

subjected to pressure, the equilibrium will be displaced in the

direction of the system that occupies the smaller volume. In

the case of a solution of a protein, hydrostatic pressure may

change the conformation, promote binding or dissociation

of a ligand, denature the protein or dissociate an oligomeric

protein [1–3]. Factors that contribute to differences in

volume between an oligomer and its subunits include

removal of packing defects, hydration of buried surfaces,

and disruption of salt bridges. As a general rule (although

there are exceptions), the pressure required to dissociate an

oligomeric protein is less than that required to denature

monomeric proteins. It therefore seems reasonable to expect

that pressure could dissociate oligomeric proteins while

having relatively little effect on the secondary and tertiary

structure of the resulting monomers. In spite of this

expectation, most monomers produced by hydrostatic

pressure have been inactive [1].

Yeast enolase (EC 4.2.1.11), which catalyzes the inter￾conversion of 2-phosphoglyceric acid and phosphoenol￾pyruvate, is a homodimer. High resolution X-ray structures

are available for the yeast enzyme [4–6], as well as enolase

from lobster [7], Escherichia coli [8] and Trypanosoma brucei

[9]. Each subunit has two domains; the larger domain is an

a/b barrel, while the smaller is a mixture of b-sheet and

a-helices. The dimer interface includes two helices in the

large domain and two b-strands in the small domain. The

active site is at the bottom of the barrel and is totally

Correspondence to M. J. Kornblatt, Department of Chemistry and

Biochemistry, Concordia University, 7141 Sherbrooke W, Montreal,

Que. H4B 1R6 Canada. Fax: +1 514 848 2868,

E-mail: [email protected]

Enzyme: enolase, EC 4.2.1.11

*Current address: Universite´ Montpellier 2, EA3763, Place Euge`ne

Bataillon, 34095 Montpellier cedex 5, France.

(Received 7 June 2004, revised 2 August 2004, accepted 6 August 2004)

Eur. J. Biochem. 271, 3897–3904 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04326.x