A modified heme-protein as a hydrolytic enzyme

Biocatalysis for the 90s LOW MOLECULAR WEIGHT THERMOSTABLE ENZYMES. M.Mattey & G.Adoga. Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow G4 0NR, Scotland. A number of proteins with a molecular weight of less than 10k but possessing enzymic activity have now been described. The extracellular esterase from Yarrowia lipolytica is typical, consisting of 56 amino acid residues of which 13 were proline.This enzyme is specific for the i position of triglycerides,requires ferric ions for maximum activity and is very thermostahle.Although the temperature optimum is 30 ° it will withstand 121 ° for 15 minutes with the loss of less than 30% of its activity. The enzyme has a hydrophobic and a hydrophilic region, the former probably binds the alkyl chain of the ester.The ferric ion is involved in the hydrolysis. Synthesis of esters occurs under conditions of low water potential. Because of their low molecular weight these enzymes are frequently overlooked, they tend to have the same relative mobility as marker dyes in electrophoresis, they are not retained by most dialysis membranes and they are poorly precipitated even in saturated ammonium sulphate.

A MODIFIED Haya

Zemel,

HEME-PROTEIN

AS

A HYDROLYTIC

A l l i e d - S i g n a l R e s e a r c h & Technology, Rd., Des Plaines, IL 6 0 0 1 7 - 5 0 1 6

J. A/~er. Chem.

Soc.

(1987),

109,

H a l o p e r o x i d a s e s are m e t a l l o e n z y m e s that catalyze the o x i d a t i o n of a halide ion by hydrogen peroxide. The oxidized halogen i n t e r m e d i a t e can h a l o g e n a t e an a p p r o p r i a t e i n t e r m e d i a t e or react with another e q u i v a l e n t of hydrogen peroxide to produce dioxygen. Three types of m e t a l l o h a l o p e r o x i d a s e s are known, including the iron-Heme, the non Heme iron and the non Heme v a n a d i u m enzymes. FeHeme h a l o p e r o x i d a s e s are found in t e r r e s t r i a l and m a r i n e organism. The novel v a n a d i u m h a l o p e r o x i d a s e is isolated from marine organisms and the newly r e p o r t e d FeNH h a l o p e r o x i d a s e has been isolated from terrestrial fungi. The general r e a c t i v i t i e s of these distinct m e t a l l o e n z y m e s are similar, although the specific metal sites appear to control the subtle differences in reactivity.

ENZYME

DNA M A N I P U L A T I O N

50 E. A l g o n q u i n

Caroline Mingins

We have b e e n e v a l u a t i n g the p o s s i b i l i t y of u t i l i z i n g existing p r o t e i n c a v i t i e s for u n n a t u r a l e n z y m a t i c catalysis. In the past i, we have shown that Myoglobin, a n o n - e n z y m a t i c protein, from which the heme has b e e n removed, functions as a true esterase. This semi-synthetic enzyme catalyzes the h y d r o l y s i s of e s t e r s and carbonates w i t h rate e n h a n c e m e n t s as high as 5000 fold. The catalysis is c h a r a c t e r i z e d by the usual enzymatic features, including saturation kinetics and stereospecificity. We have e x p l o r e d this c a t a l y t i c activity in terms of s u b s t r a t e selectivity, pH dependence, active site residues and the kinetic p a r a m e t e r s of the i n d i v i d u a l acylation and d e a c y l a t i o n steps. The results c o n f i r m that a h i s t i d i n e residue is r e s p o n s i b l e for the h y d r o l y t i c activity in the empty heme cavity. Large rate a c c e l e r a t i o n s o b s e r v e d with a v a r i e t y of s u b s t r a t e s suggest that the apoM y o g l o b i n has broad specificity. Other m e c h a n i s t i c a s p e c t s of the h y d r o l y s i s r e a c t i o n will also be presented. i. H. Zemel,

H a l o p e r o x i d a s e s from Marine and T e r r e s t r i a l Organisms: Broad Biological D i s t r i b u t i o n and Diverse Metal Composition. A. Butler, D e p a r t m e n t of Chemistry, U n i v e r s i t y of California, Santa Barbara, CA 93106

1875.

AFRC UK

S.M.

Institute

IN LOW WATER

Furniss,

SYSTEMS

A. Bryan Hanley,

of Food Research,

Colney

Alan R. Mackie

and James

Lane,

NR4

Norwich,

7UA,

Various types of nucleic acids have been s o l u b i l i s e d in organic solvents by e n c a p s u l a t i o n in reverse micelles stabilised by a range of surfactants. We have recently shown that type II r e s t r i c t i o n enzymes which normally cleave DNA at b a s e - s p e c i f i c s e q u e n c e s have m o d i f i e d activities in one such system. In an e x t e n s i o n of these studies we have examined D N A - r e s t r i c t i o n enzyme interactions in a range of other low water s u r f a c t a n t and s u r f a c t a n t - f r e e systems. We have been able to show that the cleavage of phage lambda DNA is d e p e n d e n t both on the water content of the system, the presence or absence of s u r f a c t a n t s and OD the type of s u r f a c t a n t employed. The physical properties of the system are also d e p e n d e n t upon the type of s u r f a c t a n t present. The type of s u r f a c t a n t has a severe and irreversible effect on the ability of certain r e s t r i c t i o n enzymes to operate e f f e c t i v e l y and this can be c o r r e l a t e d with the charge on the surfactant. Detailed ligation studies have shown that the restriction enzyme s p e c i f i c i t y remains unaltered. Fragments cleaved in a low water system are c o m p a t i b l e with fragments o b t a i n e d from a normal aqueous incubation. The physical c h a r a c t e r i s t i c s of the phage lambda DNA in low water systems appears to cause changes in site preference with certain cleavage sequences being inaccessible to the enzyme. These o b s e r v a t i o n s have some r e l e v a n c e to the p a c k a g i n g of DNA and its interactions in certain ~n_vivo systems.

Enzyme Microb. Technol., 1991, vol. 13, June

525