The metallobiochemistry of ribonuclease H.

METAL COMPLEXES

JO42

THE METALLOBIOCHEMISTRY

Ruwen Jou and J, A. Cowan Department of Chemistry, The USA. The biological chemistry chemical and spectroscopic sphere

complexation

interactions

Ohio

State

& DNA

OF RIBONUCLEASE

University,

Columbus,

Ohio

461

H. 43210,

of magnesium ion has been investigated by use of methods. Previous studies suggested that outer

of [Mg(H20)62+]

is a common

with RNA and metalloenzymes

coordination

[1,2]. These results suggested

state for the use

of [CO(NH&~+] as a probe of outer sphere magnesium cofactor chemistry; the coordination state of the active form of the metal is defined, and so its role in catalysis may be deduced. RNase H selectively

cleaves

the RNA strand

of an RNA-DNA

hybrid.

Both CO(NI-I&~+, and Co(en)g3+ serve to activate the enzyme, suggesting that magnesium may bind to RNase H and hybrid substrate as an outersphere complex [Mg(H20)b2+]. The relative kinetic parameters for RNase H digestion of (J3H]A.dT12o with a variety of metallocofactors have been determined. Preliminary data shows

the following

trend:

- V,ax[Mn(aq)2+] V max[Mg(aq)2+] reactivity of the “outer sphere” magnesium Mg(H2012+]

cofactor

V,,,[CO(NH~)~~+] > V,ax[Ca(aq)2+]. cobalt complexes

- V,ax[Co(en133+]

serves neither to activate a bound nucleophile

(I) nor acts as a template/Lewis

group undergoing hydrolytic cleavage. relief between the negatively charged enzyme (III). There is also a dependence

-

Most significantly, the implys that the normal [Mg(OH)+

or

acid (II) to the phosphodiestcr

Rather, Mg(aq12+ provides electrostatic substrate and the catalytic site on the on the ionic radius of the metal ion.

II

FIGURE 1. 2.

1. Possible

roles for divalent magnesium

as a nuclease metallocofactor

S. S. Reid, J. A. Cowan, J. Am. Chem. Sot, 113,673-675, (1991). Am. Chm. Sot, 113,675-676, (1991). S.S. Reid, J.A. Cowan, Biochemistry, 29, 60256032, (1990).

J. A. Cowan, 1.