E!nzymes from a `l'hermophilic bacterium, QRXOCUU fiuicamta;

Christian B. Anfinsen

  The discovery of hyperthermophilic archaebacteria has provided a valu.able tool

for the analysis of protein stability, The intrinsic thermal stability of the enzymes

isolated horn these sources makes it possible to study the molecular mechanisms

governing structure and function in a system adapted for elevated temperactutes, The

thermostability exhibited by these enzymes is maintained without any components unique

to thermophiles, suggesting that the increase in molecular stability is accomplished

through the same stereochemical interactions found in their mesophilic counterparts. The

characteristic range of activity observed in hyperthermophilic enzymes tends to parallel

growth temperature, there being little or no activity at temperatures which would be

optimal for their mesophilic counterparts. Through analysis of these enzymes it should

be possible to determine the stabilizing interactions by which the enzymes maintain

activity at extreme temperatures.

  P~~~~oc~us&&sz.u is an anaerobic marine heterotroph with an optimal growth

temperature of lOO*C, isolated by FiaIa and Stetter (1986) from solfataric mud off the

coast of Vulcan0 isiand, Italy. The a-amylase from fyrococcru fwiosz~~ has been purified

to homogeneity. The enzyme is a homodirner with a subunit molecular mass of 66 kDa.

The isoelectric point is 4.3. The enzyme displays optimal activity, with substantial

thermal stabilipi, at lCW'C, with the onset of activity at approximately 40oC Unlike

                                *+
mesophilic cr-amylases there is no dependence on Ca  for activity or thermostability.

The enzyme displays a broad range of substrate specificity, with the capacity to hydrolyze

carbohydrates as simple as maltotriose. No substrate binding occurs below the


temperature threshold of activity, and a decrease in Km accompanies an in.crease in

temperature. UV spectroscopy and intrinsic fluorescence measurements detected no

temperature-dependent structural reorganization. Hydrogen exchange results Tit&ate

that the molecule is rigid, with only a slight increase in conformational flexibility at

elevated temperature. Scanning microcalorimetry detected no considerable change in

the heat capacity function, at the pH of optimal activity, within the tempera&re range in

which activity is induced. The heat absorption peak due to denaturation, under these

conditions, occurred within the temperature range of 90-120oC. At temperatures below

90oC no excess heat absorption or change in the CD spectra were observed which could

be associated with the cooperative conformational transition of the protein.

   Out current investigations involve the isolation and characterization of two

additional enzymes from sonicates of pVrococcurfuriosus, DNase and a serine proteinase.

These enzymes also exhibit maximum activity at temperatures in the neighborhood of

100oC. Following physical characterization of these proteins as a function of

temperature, we plan to study the tertiary structure of one of the three proteins by x-ray

crystallography.